Method for treating a biologically contaminated surface

ABSTRACT

A method of preparing iron oxide nanoparticles using an herbal mixture comprising Capparis spinosa, Cichorium intybus, Solanum nigrum, Cassia occidentalis, Terminalia arjuna, Achillea millefolium, and Tamarix gallica. The method produces crystalline γ—Fe2O3 nanoparticles which are superparamagnetic. The iron oxide nanoparticles are used in a method of killing or inhibiting the growth of a bacteria and/or fungus, particularly in the form of a biofilm. The nanoparticles are also used in a method of treating colon cancer.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure relates to a method of preparing iron oxidenanoparticles with an extract of a plant mixture comprising Capparisspinosa, Cichorium intybus, Solanum nigrum, Cassia occidentalis,Terminalia arjuna, Achillea millefolium, and Tamarix gallica.

Discussion of the Background

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly nor impliedly admitted as prior art against the presentinvention.

The increasing emergence and re-emergence of antibiotic resistance inbacteria and Candida is a serious global concern for physicians,researchers and pharmaceutical industries. The infections caused bythese drug resistant organisms are very difficult to diagnose and treatand thus cause increased morbidity and mortality compared to otherinfections. Additionally, these organisms resistance to standardtreatments causes serious problems when these organisms form biofilms.Biofilms are static complex microbial communities that can grow and formon surfaces of various kinds of medical devices and implants e.g.,dental implants, catheters and sutures etc. [Sharma D, et. al.,Antimicrobial Resistance & Infection Control. 2019; 8(1):76].

Biofilms comprise packed microbial populations held together by anextra-cellular matrix which is secreted by the microbes. Theextra-cellular matrix is formed from materials such asexopolysaccharides, proteins, extracellular DNA and amyloidogenicproteins. Biofilm formation is a unique characteristic feature of anumber of microbial species such as Pseudomonas species, Staphylococcusspecies, Streptococcus species, Escherichia coli, and Candida species.Candida albicans is an opportunistic fungal pathogen and is the majorcausative agent of oropharyngeal candidiasis, especially inimmunocompromised patients [Salvatori O, et. al., Journal of dentalresearch. 2016; 95(4):365-71; and Jalal M, et. al., Internationaljournal of nanomedicine. 2019; 14:4667]. C. albicans biofilms structuresare generally composed of multiple types of cells e.g., round buddingyeast-form cells, elongated hyphal cells and oval pseudohyphal cells,which are encased in an extracellular matrix [Gulati M, et. al.,Microbes and infection. 2016 May 1; 18(5):310-21]. C. albicans is thepredominant yeast that has been isolated from medical device relatedinfections e.g., pacemakers, joint prostheses, urinary and centralvenous catheters, heart valves, contact lenses, and dentures. In theUnited States, each year more than five million central venous cathetersare placed and it has been found that biofilm infection occurs in morethan 50% of these catheters [Fox E P, & Nobile C J. The role of Candidaalbicans biofilms in human disease. In: Dietrich L A, Friedmann T S,editors. Candida albicans symptoms, causes and treatment options. NovaScience Publishers; 2013. pp. 1-24]. Biofilms provide protection to themicroorganism from various adverse environmental factors such as alteredosmolarity and pH, nutrients paucity, and mechanical and shear forces.Additionally, biofilms also block the diffusion and penetration ofantimicrobial agents inside the microbial biofilm communities. Thus, thebiofilm extracellular matrix provides additional resistance strength tomicrobes which allows them to survive not only in harsh environments,but also makes them resistant to antimicrobial drugs which may lead tothe emergence of multidrug-resistant, extensively drug-resistant, andtotally drug-resistant bacteria [Stewart P S, International journal ofmedical microbiology. 2002; 292(2):107-13]. It has been reported thatabout 80% of chronic and recurrent microbial infections in the humanbody are due to microbial biofilms. Further, it has been reported thatthe microbial cells encased in biofilms matrix were 10-1000 times moreantibiotics resistance than the planktonic cells [Mah T-F, FutureMicrobiol. 2012; 7:1061-72]. Therefore, disabling the biofilm formationby bacteria and Candida using unconventional antimicrobial agents suchas nanoparticles may be an attractive alternative approach to treat andprevent the infection caused by these pathogenic bacteria and Candidaspecies.

Recently, nanotechnology and nanomedicines have gained great attentionas antimicrobial agents to combat infection caused by biofilm-forming ordrug resistant bacteria and fungi. Significant research on antimicrobialpotential of various types of nanoparticles has been reported in theliterature against different bacterial and fungal strains including Agnanoparticles [Jalal M, et. al., International Journal of AdvancedResearch. 2016; 4(12):428-Ali S G, et. al., In Silico Pharmacology.2017; 5(1):12; and Almatroudi A, et. al., Processes. 2020; 8(4):388], Aunanoparticles [Ali S G, et. al., Antibiotics. 2020; 9(3):100], ZnOnanoparticles [Sultan A, et. al., Int. J. Curr. Microbiol. App. Sci.2015; 1:38-47; Ali S G, et. al., Antibiotics. 2020; 9(5):260; and PrasadK S, et. al., Biomolecules. 2020; 10(7):982], and Cu nanoparticles[Thiruvengadam M, et. al., Bioprocess and biosystems engineering. 2019;42(11):1769-77]. These nanoparticles, however, are not withoutdrawbacks. For example, while Ag nanoparticles have shown antimicrobialactivity against a large number of bacterial and fungal species, theyhave also been shown to exhibit toxicity in zebrafish [Asharani PV, et.al., Nanotechnology. 2008; 19(25):255102], Crucian carp, Eurasian perch[Bilberg K, et. al., Aquat Toxicol. 2011; 104(1):145-52], in varioushuman cell lines [Kawata K, et. al., Environ Sci Technol. 2009;43(15):6046-51; and Foldbjerg R, et. al., Arch Toxicol. 2011;85(7):743-50] and in vivo in mice [Ansari M A, et. al., Environmentaltoxicology. 2016; 31(8):945-56].

Therefore, an ideal microbiocidal agent should be toxic to bacteria andfungi, but safe to human cells. One such candidate is iron and itscompounds. Iron oxide NPs (IONPs) have been shown to be non-toxic[Samanta B, et. al., J Mater Chem. 2008; 18(11):1204-8; Sun C, et. al.,ACS Nano. 2010; 4(4):2402-10; and Prodan A M, et. al., J Nanomater.2013; 2013:587021]. Further, iron oxide nanoparticle can degraded bynatural body processes and can act as a supplementary iron source[Weissleder R, et. al., Am J Roentgenol. 1989; 152(1):167-73]. The IONPshave been shown to inhibit growth of Staphylococcus aureus, Escherichiacoli [Darwish M S A, et. al., J Nanomater. 2015; 2015:416012], Bacillussubtillis and P. aeruginosa [Farouk F, et. al., Biotechnology Letters.2020; 42(2):231-40], prevent biofilm formation by P. aeruginosa [ArmijoL M, et. al., Journal of Nanobiotechnology. 2020; 18(1):1-27] and S.aureus [Shi S F, et. al., International journal of nanomedicine. 2016;11:6499].

While many methods have been used for synthesizing iron oxidenanoparticles, “green” approaches for NPs synthesis have severaladvantages: they are eco-friendly, cost-effective, facile, non-toxic,and rapid and most importantly additional chemical capping andstabilization agents not required. One such green approach is the use ofplants or plant extracts as reducing or stabilizing/capping agents fornanoparticles. Additionally, many herbal plants, their parts, and theirproducts have themselves been used for the treatment of various kinds ofdiseases.

Accordingly, the present disclose describes a method of preparing ironoxide nanoparticles using an extract of a plant mixture. The methodproduces iron oxide nanoparticles which may be stabilized or capped byphytochemicals which are present in the plant mixture. These iron oxidenanoparticles with the phytochemical stabilizing/capping agents areuseful as antimicrobial agents and in colon cancer treatment.

SUMMARY OF THE INVENTION

The present disclosure relates to a method of preparing iron oxidenanoparticles, the method comprising mixing an iron precursor solutioncomprising an iron (III) salt and a solvent with an extract of a plantmixture to form a reaction mixture, heating the reaction mixture to formthe iron oxide nanoparticles, and isolating the iron oxidenanoparticles, wherein the plant mixture comprises Capparis spinosa,Cichorium intybus, Solanum nigrum, Cassia occidentalis, Terminaliaarjuna, Achillea millefolium, and Tamarix gallica.

In some embodiments, the method further comprises soaking the plantmixture in water in an amount of 1 g of plant mixture per 1 to 25 mL ofwater at 5 to 50° C. for 4 to 48 hours to form a plant suspension, andfiltering the plant suspension to form the extract.

In some embodiments, the plant mixture comprises 26 to 27.5 wt %Capparis spinosa, 26 to 27.5 wt % Cichorium intybus, 12.5 to 14 wt %Solanum nigrum, 6 to 7 wt % Cassia occidentalis, 12.5 to 14 wt %Terminalia arjuna, 6 to 7 wt % Achillea millefolium, and 6 to 7 wt %Tamarix gallica.

In some embodiments, the solvent is water, the iron (III) salt is aniron (III) halide, and the heating is performed at 40 to 80° C. for 15to 180 minutes. In some embodiments, the reaction mixture has an iron(III) concentration of 0.25 to 1.25 mM and the extract is present in thereaction mixture in an amount of 24 to 120 mL extract per mmol of iron(III).

In some embodiments, the iron oxide nanoparticles comprise crystallineγ-Fe₂O₃ by PXRD and a mean particle size of 10 to 100 nm by electronmicroscopy.

In some embodiments, the iron oxide nanoparticles have a saturationmagnetization of 17.5 to 27.5 emu/g and a coercivity less than 250 Oe at275 to 325 K.

The present disclosure also relates to iron oxide nanoparticles,comprising iron oxide stabilized with an extract of a plant mixturecomprising Capparis spinosa, Cichorium intybus, Solanum nigrum, Cassiaoccidentalis, Terminalia arjuna, Achillea millefolium, and Tamarixgallica.

In some embodiments, the iron oxide nanoparticles comprise crystallineγ-Fe₂O₃ by PXRD.

In some embodiments, the iron oxide nanoparticles have a mean particlesize of 10 to 100 nm by electron microscopy.

In some embodiments, the iron oxide nanoparticles have a saturationmagnetization of 17.5 to 27.5 emu/g and a coercivity less than 250 Oe at275 to 325 K.

In some embodiments, the plant mixture comprises 26 to 27.5 wt %Capparis spinosa, 26 to 27.5 wt % Cichorium intybus, 12.5 to 14 wt %Solanum nigrum, 6 to 7 wt % Cassia occidentalis, 12.5 to 14 wt %Terminalia arjuna, 6 to 7 wt % Achillea millefolium, and 6 to 7 wt %Tamarix gallica.

In some embodiments, the extract comprises at least three selected fromthe group consisting of: n-hexadecanoic acid, (Z,Z)-9,12-octadecadienoicacid, (Z)-9-octadecenoic acid, octadecanoic acid,(Z)-3-(pentadec-8-en-1-yl)phenol, piperine,2-(hydroxymethyl)-2-nitro-1,3-propanediol, and tetradecanoic acid.

In some embodiments, the extract further comprises at least one selectedfrom the group consisting of: quercetin, kaempferol, cappariloside A,capparine A, capparine B, capparisine A, capparisine B, capparisine C,lactucin, lactucopicrin, aesculetin, aesculin, cichoriin, umbelliferone,scopoletin, 6,7-dihydrocoumarin, solasodine, solanine, emodin,cassiollin, cassia occidentanol I, cassia occidentanol II, arjunin,arjunic acid, arjungenin, arjunetin, arjunone, arjunoside I, arjunosideII, arjunoside III, arjunoside IV, archilletin, achilleine, apigenin,luteolin, tamarixin, tamarixetin, 4-methylcoumarin, and troupin.

The present disclosure also relates to a method of killing or inhibitingthe growth of bacteria and/or fungus, the method comprising exposing thebacteria and/or fungus to the iron oxide nanoparticles.

In some embodiments, the bacteria and/or fungus is in the form of abiofilm.

In some embodiments, the bacteria and/or fungus is at least one selectedfrom the group consisting of P. aeruginosa, S. aureus, and C. albicans.

In some embodiments, the iron oxide nanoparticles have a minimuminhibitory concentration (MIC) for P. aeruginosa of 0.60 to 1.5 mg ironoxide nanoparticles per mL, a MIC for S. aureus of 0.9 to 2.45 mg ironoxide nanoparticles per mL, and a MIC for C. albicans of 1.30 to 2.85 mgiron oxide nanoparticles per mL.

The present disclosure also relates to a method of treating coloncancer, the method comprising administering to a patient in need oftherapy an effective dose of the iron oxide nanoparticles.

In some embodiments, the iron oxide nanoparticles are administered in anamount sufficient to provide a concentration of 15 to 200 μg iron oxidenanoparticles per mL of tumor volume at a colon cancer-containing site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows UV-vis spectra of the extract of the plant mixture and thesynthesized iron oxide nanoparticles;

FIG. 2 shows FT-IR spectra of the extract of the plant mixture and thesynthesized iron oxide nanoparticles;

FIG. 3A shows a typical GC-MS chromatogram of the iron oxidenanoparticles synthesized using the extract of the plant mixtureindicating 37 peaks of extract phytochemicals associated with thenanoparticles;

FIG. 3B shows a bar graph showing the major phytochemicals present inthe extract;

FIGS. 4A-4D show electron microscopy characterization of the synthesizediron oxide nanoparticles where FIG. 4A is a scanning electron microscopyimage of the iron oxide nanoparticles, FIG. 4B is a transmissionelectron microscopy image of the iron oxide nanoparticles, FIG. 4C is aselected area EDX spectrum of the iron oxide nanoparticles, and FIG. 4Dis a size histogram of the iron oxide nanoparticles derived from theelectron microscopy images;

FIG. 5 shows a PXRD pattern of the iron oxide nanoparticles;

FIG. 6 is a plot of the magnetization vs applied field for the ironoxide nanoparticles;

FIGS. 7A-7F are scanning electron microscopy images of bacteria andfungus where FIG. 7A shows P. aeruginosa before exposure to the ironoxide nanoparticles, FIG. 7B shows P. aeruginosa after exposure to theiron oxide nanoparticles, FIG. 7C shows MRSA before exposure to the ironoxide nanoparticles, FIG. 7D shows MRSA after exposure to the iron oxidenanoparticles, FIG. 7E shows C. albicans before exposure to the ironoxide nanoparticles, and FIG. 7F shows C. albicans after exposure to theiron oxide nanoparticles;

FIG. 8 is a bar graph depicting the dose dependent inhibition of biofilmformation for P. aeruginosa, MRSA and C. albicans exposed to the ironoxide nanoparticles;

FIGS. 9A-9F are scanning electron microscopy images of biofilms ofbacteria and fungus where FIG. 9A shows a biofilm of P. aeruginosabefore exposure to the iron oxide nanoparticles, FIG. 9B shows a biofilmof P. aeruginosa after exposure to the iron oxide nanoparticles, FIG. 9Cshows a biofilm of MRSA before exposure to the iron oxide nanoparticles,FIG. 9D shows a biofilm of MRSA after exposure to the iron oxidenanoparticles, FIG. 9E shows a biofilm of C. albicans before exposure tothe iron oxide nanoparticles, and FIG. 9F shows a biofilm of C. albicansafter exposure to the iron oxide nanoparticles;

FIG. 10 is a graph of MTT cell viability analysis of HCT-116 cells aftertreatment with the iron oxide nanoparticles at concentrations of 0, 10,50, and 100 μg/mL; and

FIGS. 11A-11D are light microscopy images showing the morphology ofHCT-116 cells 72 h after exposure to the iron oxide nanoparticles atconcentrations of 0, 10, 50 and 100 μg/mL as well as arrows indicatingcell dead cell debris.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, it is understood that other embodimentsmay be utilized and structural and operational changes may be madewithout departure from the scope of the present embodiments disclosedherein.

Definitions

As used herein the words “a” and “an” and the like carry the meaning of“one or more.”

As used herein, the terms “optional” or “optionally” means that thesubsequently described event(s) can or cannot occur or the subsequentlydescribed component(s) may or may not be present (e.g., 0 wt. %).

According to a first aspect, the present disclosure relates to a methodof preparing iron oxide nanoparticles, the method comprising mixing aniron precursor solution comprising an iron (III) salt and a solvent withan extract of a plant mixture to form a reaction mixture, heating thereaction mixture to form the iron oxide nanoparticles, and isolating theiron oxide nanoparticles. The plant mixture used in this methodcomprises Capparis spinosa (also referred to as caper bush and Findersrose), Cichorium intybus (also referred to as chicory or commonchicory), Solanum nigrum (also referred to as European black nightshade,black nightshade, and blackberry nightshade), Cassia occidentalis (alsoreferred to as senna occidentalis, 'au'auko'i, septicweed, coffee senna,coffeeweed, Mogdad coffee, negro-coffee, senna coffee, Stephanie coffee,stinkingweed, styptic weed, and bana chakunda) , Terminalia arjuna (alsoreferred to as arjuna or Arjun tree), Achillea millefolium (alsoreferred to as yarrow or common yarrow), and Tamarix gallica (alsoreferred to as French tamarisk). The plant mixture is also referred toas Liv52™ available from The Himalaya Drug Company (Himalaya).

In general, the plant mixture may contain the plants listed above in anyrelative amounts. In preferred embodiments, the plant mixture comprises26 to 27.5 wt %, preferably 26.5 to 27.25 wt %, preferably 26.75 to 27.0wt %, preferably 26.8 to 26.9 wt % Capparis spinosa, 26 to 27.5 wt %,preferably 26.5 to 27.25 wt %, preferably 26.75 to 27.0 wt %, preferably26.8 to 26.9 wt % Cichorium intybus, 12.5 to 14 wt %, preferably 12.75to 13.75 wt %, preferably 12.8 to 13.7 wt %, preferably 12.9 to 13.6 wt%, preferably 13 to 13.5 wt %, preferably 13.1 to 13.4 wt %, preferably13.2 to 13.3 wt % Solanum nigrum, 6 to 7 wt %, preferably 6.25 to 6.9 wt%, preferably 6.4 to 6.8 wt %, preferably 6.6 to 6.7 wt % Cassiaoccidentalis, 12.5 to 14 wt %, preferably 12.75 to 13.75 wt %,preferably 12.8 to 13.7 wt %, preferably 12.9 to 13.6 wt %, preferably13 to 13.5 wt %, preferably 13.1 to 13.4 wt %, preferably 13.2 to 13.3wt % Terminalia arjuna, 6 to 7 wt %, preferably 6.25 to 6.9 wt %,preferably 6.4 to 6.8 wt %, preferably 6.6 to 6.7 wt % Achilleamillefolium, and 6 to 7 wt %, preferably 6.25 to 6.9 wt %, preferably6.4 to 6.8 wt %, preferably 6.6 to 6.7 wt % Tamarix gallica.

Liv 52 is a polyherbal ayurvedic medicinal formulation of Capparisspinosa (130 mg), Cichorium intybus (130 mg), Solanum nigrum (64 mg),Cassia occidentalis (32 mg), Terminalia arjuna (64 mg), Achilleamillefolium (32 mg), and Tamarix gallica (32 mg) plants, and is mostcommonly prescribed as a traditional hepatotonic for the treatment ofliver cirrhosis and viral hepatitis in India. The formulation is usedto, for example, stimulate appetite and protect the liver againsthepatotoxins (i.e., beryllium, CC14, paracetamol, alcohol) [De Silva HA, et. al., J Ethnopharmacol.2003; 84(1):47-50]. Further, herbs andherbal mixture such as Liv 52 possess large amounts of polyphenolic andlong-chain saturated and unsaturated fatty acid which may act asreducing, stabilising and capping agents for nanoparticles.

In general, any part or combination of parts of the plants listed abovemay be used in the extract used in the current invention. For example,the extract may be made using whole plants, roots, stems, leaves,flowers, bark, bulbs, fruits, seeds, buds, or any combination thereof.In some embodiments, the extract of the plant mixture comprises a wholeplant extract of one or any combination of the plants listed above. Insome embodiments, the extract of the plant mixture comprises a rootextract of one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a stem extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a leaf extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a flower extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a bark extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a bulb extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a fruit extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a seed extractof one or any combination of the plants listed above. In someembodiments, the extract of the plant mixture comprises a bud extract ofone or any combination of the plants listed above. In general, the plantmixture may be prepared for making the extract by any suitable techniqueknown to one of ordinary skill in the art. For example, the plantmixture or any component thereof may be dried before making the extractand/or reduced in size to small particles. In general, the plant mixtureor any component thereof may be reduced to small particles using anysuitable technique known to one of ordinary skill in the art. Examplesof such techniques include, but are not limited to milling, grinding,ball milling, chopping, pulverizing, crushing, pounding, mincing,shredding, smashing, and fragmenting. In some embodiments, the reducingto small particles may take place using a mill, ball mill, rod mill,autogenous mill, semi-autogenous grinding mill, pebble mill, buhrstonemill, burr mill, tower mill, vertical shaft impactor mill, a low energymilling machine, grinder, pulverizer, mortar and pestle, blender,crusher, or other implement used to reduce a material to smallparticles.

In general, the extract of the plant mixture may be prepared by anysuitable method known to one of ordinary skill in the art. Such a methodmay involve, for example, plant tissue homogenization, soaking,maceration, digestion, decoction, infusion, percolation, Soxhletextraction, superficial extraction, ultrasound-assisted,microwave-assisted extraction, or any combination thereof. In someembodiments, the plant mixture is prepared by soaking. The soaking mayor may not involve agitation, such as shaking or stirring. In general,any suitable solvent known to one of ordinary skill in the art may beused to prepare the extract of the plant mixture. Examples of suchsuitable solvents include, but are not limited to hexane, petroleumether, diethyl ether, ethyl acetate, chloroform, dichloromethane,acetone, n-butanol, isopropanol, n-propanol, ethanol, methanol, water,and mixtures thereof. In some embodiments, the solvent comprises water.In preferred embodiments, the solvent is water. In general, the plantmixture may be used in any suitable amount known to one of ordinaryskill in the art to prepare the extract. In some embodiments, theextract is prepared at a concentration (which may be measured in theamount of plant mixture per volume of solvent) at which it is intendedto be used. In alternative embodiments, the extract is not prepared at aconcentration at which it is intended to be used. In such embodiments, aconcentration of the extract may be adjusted before use in the method ofpreparing iron oxide nanoparticles. Such adjustment may be made by anysuitable method known to one of ordinary skill in the art. In someembodiments, the extract is diluted to a lower concentration compared toa preparation concentration for use in the method of preparing ironoxide nanoparticles. In alternative embodiments, the extract isconcentrated, for example by evaporation, to a higher concentrationcompared to the preparation concentration for use in the method ofpreparing iron oxide nanoparticles. In some embodiments, the extract isprepared using 1 g of plant mixture per 1 to 25 mL, preferably 2.5 to 20mL, preferably 5 to 15 mL, preferably 7.5 to 12.5 mL, preferably 8 to 12mL, preferably 9 to 11 mL, preferably 10 mL of solvent. In someembodiments, the soaking is performed at 5 to 50° C., preferably 10 to40° C., preferably 15 to 35° C., preferably 20 to 30° C., preferably22.5 to 27.5° C., preferably about 25° C. In some embodiments, thesoaking is performed for 4 to 48 hours, preferably 6 to 44 hours,preferably 8 to 40 hours, preferably 10 to 36 hours, preferably 12 to 32hours, preferably 14 to 28 hours, preferably 16 to 24 hours. Thissoaking creates a plant suspension which comprises a liquid solventextract and suspended plant solids. In preferred embodiments, the plantsolids are removed following the soaking. In general, the plant solidsmay be removed by any suitable technique known to one of ordinary skillin the art. Examples of such suitable techniques include, but are notlimited to decantation, centrifugation, and filtration, but excludingtechniques such as evaporation and distillation. In preferredembodiments, the method further comprises soaking the plant mixture inwater in an amount of 1 g of plant mixture per 1 to 25 mL, preferably2.5 to 20 mL, preferably 5 to 15 mL, preferably 7.5 to 12.5 mL,preferably 8 to 12 mL, preferably 9 to 11 mL, preferably 10 mL of waterat 5 to 50° C., preferably 10 to 40° C., preferably 15 to 35° C.,preferably 20 to 30° C., preferably 22.5 to 27.5° C., preferably about25° C. for 4 to 48 hours, preferably 6 to 44 hours, preferably 8 to 40hours, preferably 10 to 36 hours, preferably 12 to 32 hours, preferably14 to 28 hours, preferably 16 to 24 hours to form a plant suspension,and filtering the plant suspension to form the extract.

In general, the iron (III) salt may be any suitable iron (III) saltknown to one of ordinary skill in the art. Examples of such suitableiron (III) salts include, but are not limited to iron (III) nitrate,iron (III) acetate, iron (III) halides including iron (III) chloride,iron (III) bromide, and iron (III) iodide, iron (III) sulfate, iron(III) oxalate, iron (III) phosphate, iron (III) gluconate, iron (III)fumarate, iron (III) citrate, and iron (III) chromate. In preferredembodiments, the iron (III) salt is an iron (III) halide. In someembodiments, the iron (III) halide is iron (III) chloride. In general,the solvent may be any suitable solvent known to one of ordinary skillin the art. Examples of such suitable solvents include but are notlimited to hexane, petroleum ether, diethyl ether, ethyl acetate,chloroform, dichloromethane, acetone, n-butanol, isopropanol,n-propanol, ethanol, methanol, water, acetaldehyde, acetic acid,acetonitrile, 1,2-butanediol, 1,3-butanediol, 1,4-nutanediol,2-butoxyethanol, butyric acid, diethanolamine, diethylenetriamine,dimethylformamide, dimethoxyethane, dimethyl sulfoxide, 1,4-dioxane,ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol,methyl diethanolamine, methyl isocyanide, N-methyl-2-pyrrolidone,1,3-propanediol, 1,5-pentanediol, propanoic acid, propylene glycol,pyridine, tetrahydrofuran, triethylene glycol, diglyme, and mixturesthereof. In some embodiments, the solvent comprises water. In someembodiments, the solvent is water. In some embodiments, the heating isperformed at 40 to 80° C., preferably 50 to 75° C., preferably 60 to 70°C., preferably 65° C. In some embodiments, the heating is performed for15 to 180 minutes, preferably 30 to 120 minutes, preferably 45 to 90minutes, preferably 50 to 70 minutes, preferably 55 to 65 minutes,preferably 60 minutes. Following the heating, the iron oxidenanoparticles may be collected or isolated. In general, the iron oxidenanoparticles may be collected or isolated by any suitable techniqueknown to one of ordinary skill in the art. Examples of such techniquesinclude, but are not limited to, liquid-liquid extraction, dialysis,centrifugation, chromatography, precipitation, filtration, anddecantation. In some embodiments, the collecting or isolating compriseswashing. In general, the washing may be performed using any suitabletechnique known to one of ordinary skill in the art. In someembodiments, the washing is performed with a wash solvent which may beany suitable solvent as described above. In some embodiments, multiplerounds of washing are performed. These multiple rounds may be performedwith the same wash solvent or with different wash solvents. In someembodiments, the solvent is water, the iron (III) salt is an iron (III)halide, and the heating is performed at 40 to 80° C. for 15 to 180minutes.

In some embodiments, the reaction mixture has an iron (III)concentration of 0.25 to 1.25 mM, preferably 0.3 to 1.2 mM, preferably0.4 to 1.1 mM, preferably 0.5 to 1.0 mM, preferably 0.55 to 0.9 mM,preferably 0.6 to 0.8 mM, preferably 0.65 to 0.75 mM. In someembodiments, extract is present in the reaction mixture in an amount of24 to 120 mL extract per mmol of iron (III), preferably 25 to 100 mL,preferably 30 to 75 mL, preferably 32.5 to 60 mL, preferably 35 to 55mL, preferably 37.5 to 50 mL, preferably 40 to 45 mL, preferably 41 to44, preferably 42 to 43 mL extract per mmol of iron (III). In someembodiments, the reaction mixture has an iron (III) concentration of0.25 to 1.25 mM and the extract is present in the reaction mixture in anamount of 24 to 120 mL extract per mmol of iron (III).

In some embodiments, the reaction mixture further comprises asupplementary reducing agent. In general, the supplementary reducingagent may be any suitable reducing agent known to one of ordinary skillin the art. Examples of reducing agents include, but are not limited toborohydrides, citrates, ascorbates, amines such as 4-aminophenol,oleylamine, trimethylamine, and indole, amino acids such as glycine,tryptophan, and proline, and hydrogen. In preferred embodiments, thereaction mixture is devoid of a supplementary reducing agent.

In some embodiments, the iron oxide nanoparticles are crystalline byPXRD. In some embodiments, the iron oxide nanoparticles comprisecrystalline γ-Fe₂O₃ by PXRD. In some embodiments, the iron oxidenanoparticles further comprise other elements besides iron and oxygenwhich are incorporated into the crystalline γ-Fe₂O₃ or an amorphousphase associated with the iron oxide nanoparticles. When such elementsare incorporated into the crystalline γ-Fe₂O₃, they may be referred toas “dopants”. In such embodiments, such elements are preferably presentin an amount of less than 10 atom %, preferably less than 7.5 atom %,preferably less than 5 atom %, preferably less than 2.5 atom %,preferably less than 1 atom %, preferably less than 0.5 atom %,preferably less than 0.1 atom %, based on a total number of iron atoms.In preferred embodiments, the iron oxide nanoparticles are devoid ofcrystalline phases which are not γ-Fe₂O₃, measured by PXRD.

In general, the iron oxide nanoparticles can be any shape known to oneof ordinary skill in the art. Examples of suitable shapes the iron oxidenanoparticles may take include spheres, spheroids, lentoids, ovoids,solid polyhedra such as tetrahedra, cubes, octahedra, icosahedra,dodecahedra, rectangular prisms, triangular prisms (also known asnanotriangles), nanoplatelets, nanodisks, rods (also known as nanorods),blocks, flakes, discs, granules, angular chunks, and mixtures thereof.In the case of nanorods, the rod shape may be defined by a ratio of arod length to a rod width, the ratio being known as the aspect ratio.For iron oxide nanoparticles of the current invention, nanorods shouldhave an aspect ratio less than 1000, preferably less than 750,preferably less than 500, preferably less than 250, preferably less than100, preferably less than 75, preferably less than 50, preferably lessthan 25. Nanorods having an aspect ratio greater than 1000 are typicallyreferred to as nanowires and are not a shape that the iron oxidenanoparticles are envisioned as having in any embodiments. In preferredembodiments, the iron oxide nanoparticles are substantially spherical.Spherical particles may be described by a measure known as sphericity.Sphericity is a measure of how closely the shape of an object resemblesthat of a perfect sphere and may be calculated for a particle by takingthe ratio of the surface area of a sphere having a volume equal to thatof the particle to the surface area of the particle. A perfect spherehas a sphericity of 1. In some embodiments, the iron oxide nanoparticleshave a mean sphericity of at least 0.75, preferably at least 0.775,preferably at least 0.80, preferably at least 0.825, preferably at least0.85, preferably at least 0.875, preferably at least 0.90, preferably atleast 0.925, preferably at least 0.95, preferably at least 0.975.

In some embodiments, the iron oxide nanoparticles have a uniform shape.Alternatively, the shape may be non-uniform. As used herein, the term“uniform shape” refers to an average consistent shape that differs by nomore than 10%, by no more than 5%, by no more than 4%, by no more than3%, by no more than 2%, by no more than 1% of the distribution of ironoxide nanoparticles having a different shape. As used herein, the term“non-uniform shape” refers to an average consistent shape that differsby more than 10% of the distribution of iron oxide nanoparticles havinga different shape. In one embodiment, the shape is uniform and at least90% of the iron oxide nanoparticles are spherical or substantiallycircular, and less than 10% are polygonal. In another embodiment, theshape is non-uniform and less than 90% of the iron oxide nanoparticlesare spherical or substantially circular, and greater than 10% arepolygonal.

In some embodiments, the iron oxide nanoparticles have a mean particlesize of 10 to 100 nm, preferably 12.5 to 75 nm, preferably 15 to 60 nm,preferably 17.5 to 50 nm, preferably to 40 nm, preferably 22.5 to 37.5nm, preferably 25 to 35 nm. In embodiments where the iron oxidenanoparticles are spherical, the particle size may refer to a particlediameter. In embodiments where the iron oxide nanoparticles arepolyhedral, the particle size may refer to the diameter of acircumsphere. In some embodiments, the particle size refers to a meandistance from a particle surface to particle centroid or center of mass.In alternative embodiments, the particle size refers to a maximumdistance from a particle surface to a particle centroid or center ofmass. In some embodiments where the iron oxide nanoparticles have ananisotropic shape such as nanorods, the particle size may refer to alength of the nanorod, a width of the nanorod, or an average of thelength and width of the nanorod. In some embodiments, the particle sizerefers to the diameter of a sphere having an equivalent volume as theparticle.

In some embodiments, the iron oxide nanoparticles are monodisperse,having a coefficient of variation or relative standard deviation,expressed as a percentage and defined as the ratio of the particle sizestandard deviation (σ) to the particle size mean (μ) multiplied by 100of less than 25%, preferably less than 10%, preferably less than 8%,preferably less than 6%, preferably less than 5%, preferably less than4%, preferably less than 3%, preferably less than 2%. In someembodiments, the iron oxide nanoparticles of the present disclosure aremonodisperse having a particle size distribution ranging from 80% of theaverage particle size to 120% of the average particle size, preferably90-110%, preferably 95-105% of the average particle size. In someembodiments, the iron oxide nanoparticles are not monodisperse.

In general, the particle size may be determined by any suitable methodknown to one of ordinary skill in the art. In some embodiments, theparticle size is determined by powder X-ray diffraction (PXRD). UsingPXRD, the particle size may be determined using the Scherrer equation,which relates the full-width at half-maximum (FWHM) of diffraction peaksto the size of regions comprised of a single crystalline domain (knownas crystallites) in the sample. In some embodiments, the iron oxidenanoparticles have a mean crystallite size of 10 to 100 nm, preferably12.5 to 75 nm, preferably 15 to 60 nm, preferably 17.5 to 50 nm,preferably 20 to 40 nm, preferably 25 to 35 nm, preferably 27.5 to 30nm, preferably 28 to 29 nm. In some embodiments, the crystallite size isthe same as the particle size. For accurate particle size measurement byPXRD, the particles should be crystalline, comprise only a singlecrystal, and lack non-crystalline portions. Typically, the crystallitesize underestimates particle size compared to other measures due tofactors such as amorphous regions of particles, the inclusion ofnon-crystalline material on the surface of particles such as bulkysurface ligands, and particles which may be composed of multiplecrystalline domains. In some embodiments, the particle size isdetermined by dynamic light scattering (DLS). DLS is a technique whichuses the time-dependent fluctuations in light scattered by particles insuspension or solution in a solvent, typically water to measure a sizedistribution of the particles. Due to the details of the DLS setup, thetechnique measures a hydrodynamic diameter of the particles, which isthe diameter of a sphere with an equivalent diffusion coefficient as theparticles. The hydrodynamic diameter may include factors not accountedfor by other methods such as non-crystalline material on the surface ofparticles such as surface ligands, amorphous regions of particles, andsurface ligand-solvent interactions. Further, the hydrodynamic diametermay not accurately account for non-spherical particle shapes. DLS doeshave an advantage of being able to account for or more accurately modelsolution or suspension behavior of the particles compared to othertechniques. In preferred embodiments, the particle size is determined byelectron microscopy techniques such as scanning electron microscopy(SEM) or transmission electron microscopy (TEM).

In some embodiments, the iron oxide nanoparticles have a saturationmagnetization of 17.5 to 27.5 emu/g, preferably 18 to 25 emu/g,preferably 19 to 24 emu/g, preferably 20 to 23 emu/g, preferably 21 to22 emu/g, preferably 21.5 emu/g. In some embodiments, the iron oxidenanoparticles have a coercivity less than 250 Oe, preferably less than225 Oe, preferably less than 200 Oe, preferably less than 175 Oe,preferably less than 150 Oe, preferably less than 125 Oe, preferablyless than 100 Oe, preferably less than 75 Oe, preferably less than 50Oe, preferably less than 25 Oe at 275 to 325 K, preferably 280 to 320 K,preferably 290 to 310 K. In preferred embodiments, the iron oxidenanoparticles are superparamagnetic at 0 to 50° C., preferably 10 to 40°C., preferably 15 to 38° C., preferably 20 to 30° C. In someembodiments, the iron oxide nanoparticles are superparamagnetic at roomtemperature. A magnetic nanomaterial may be characterized by itsblocking temperature, the temperature at which the magnetic behavior ofthe material changes from superparamagnetic toferromagnetic/ferromagnetic. In some embodiments, the iron oxidenanoparticles have a blocking temperature below 20° C., preferably below15° C., preferably below 10° C., preferably below 5° C., preferablybelow 0° C., preferably below −10° C., preferably below −25° C.,preferably below −50° C., preferably below −78° C., preferably below−100° C. The magnetic ordering in nanomaterial can be affected byfactors such as the composition and particle size. In general, the ironoxide may be of any suitable composition and/or particle size so as toremain superparamagnetic at temperatures at 0 to 50° C., preferably 10to 40° C., preferably 15 to 38° C., preferably 20 to 30° C.

In some embodiments, the extract comprises at least three selected fromthe group consisting of: n-hexadecanoic acid, (Z,Z)-9,12-octadecadienoicacid, (Z)-9-octadecenoic acid, octadecanoic acid,(Z)-3-(pentadec-8-en-1-yl)phenol, piperine,2-(hydroxymethyl)-2-nitro-1,3-propanediol, and tetradecanoic acid. Insome embodiments, the extract further comprises at least four selectedfrom the group above. In some embodiments, the extract further comprisesat least five selected from the group above. In some embodiments, theextract further comprises at least six selected from the group above. Insome embodiments, the extract further comprises at least seven selectedfrom the group above. In some embodiments, the extract further comprisesat least eight selected from the group above. In some embodiments, theextract further comprises all of the members of the group above.

In some embodiments, the extract further comprises at least one selectedfrom the group consisting of: quercetin, kaempferol, cappariloside A,capparine A, capparine B, capparisine A, capparisine B, capparisine C,lactucin, lactucopicrin, aesculetin, aesculin, cichoriin, umbelliferone,scopoletin, 6,7-dihydrocoumarin, solasodine, solanine, emodin,cassiollin, cassia occidentanol I, cassia occidentanol II, arjunin,arjunic acid, arjungenin, arjunetin, arjunone, arjunoside I, arjunosideII, arjunoside III, arjunoside IV, archilletin, achilleine, apigenin,luteolin, tamarixin, tamarixetin, 4-methylcoumarin, and troupin. In someembodiments, the extract further comprises at least two selected fromthe group above. In some embodiments, the extract further comprises atleast three selected from the group above. In some embodiments, theextract further comprises at least four selected from the group above.In some embodiments, the extract further comprises at least fiveselected from the group above. In some embodiments, the extract furthercomprises at least six selected from the group above. In someembodiments, the extract further comprises at least seven selected fromthe group above. These chemicals, as well as others not named here,which are present in the extract of the plant mixture may be referred tocollectively as “extract phytochemicals”.

In some embodiments, the extract phytochemicals act as surface ligandsfor the iron oxide nanoparticles. In some embodiments, the extractphytochemicals act as surface ligands by binding non-oxidatively to asurface of the iron oxide nanoparticles. Such non-oxidative binding mayoccur through, for example, non-deprotonated alcohol, ether, amine,amide, carboxyl, carbonyl, thiol, disulfide, ester, or other functionalgroup acting as an “L-type” ligand and/or physisorption, This binding isdistinct from oxidative binding seen in, for example, carboxylates,alkoxides, hydroxide ions or halides, which may act as “X-type” ligands.The non-oxidative binding may occur through metal-ligand coordinationtype interactions between appropriate functional groups on the extractphytochemicals. The alcohol groups should exist in alcohol form, thatis, bearing the hydroxyl proton. Such a form is distinct from thedeprotonated alkoxide form. Additionally, there may be non-chemicalinteractions which cause physisorption of the extract phytochemicals tothe surface of the iron oxide nanoparticle. Examples of suchnon-chemical interactions include electrostatic interactions such as ion(or charged species in general)-ion interactions, ion-dipoleinteractions, or dipole-dipole interactions; and Van der Waalsinteractions. While the surface of the iron oxide nanoparticle may havea charge, the extract phytochemicals may be present in either charged oruncharged form. The binding of the extract phytochemicals may also occurionically or oxidatively. Such oxidative binding may occur, for example,through or involving the formation of, surface iron atoms formally inthe +3 oxidation state but which are not fully incorporated into thecrystalline γ-Fe₂O₃ or an amorphous iron oxide phase which may bepresent on the surface of the iron oxide nanoparticle or through aligand which is acting as an “X-type” ligand. An example of suchoxidative binding is through a thiolate, alkoxide, or amide ion (adeprotonated amine derivative not to be confused with the organicfunctional group commonly depicted as —C(O)NR2).

In some embodiments, the iron oxide nanoparticles further comprisesurface ligands which are not present in the extract. In general, thesurface ligands may be any suitable surface ligands known to one ofordinary skill in the art. Examples of such surface ligands include, butare not limited to carboxylates (often referred to by their acid forms)such as citrate (citric acid), oleate (oleic acid), amines such asoleylamine, hexadecylamine, octadecylamine, and 1,6-diaminohexane;thiols such as decanethiol, dodecanethiol, and thiol-terminatedpolyethylene glycol (PEG-SH); lipids, proteins such as albumin,ovalbumin, thrombin, and lactoglobulin, polysaccharides such as chitosanand dextran; phosphines such as trioctylphosphine, trioctylphosphineoxide, and triphenylphosphine; and surfactants such ascetyltrimethylammonium bromide (CTAB). For examples of surface ligands(also called capping ligands or capping agents), see Javed, et. al.,Kobayashi, et. al., and Guerrini, et. al. [Javed, R., et. al., Journalof Nanobiotechnology, 2020, 18, article number 172; Kobayashi, K., et.al., Polymer Journal, 2014, 46, 460-468; and Guerrini, L., et. al.,Materials, 2018, 11, 1154].

In some embodiments, the iron oxide nanoparticles have a coating. Insuch embodiments, the iron oxide nanoparticles should be understood tocomprise an iron oxide portion and a coating portion. That is, thecoating forms an integral part of the iron oxide nanoparticles. Inembodiments where the iron oxide nanoparticles have a coating, the“surface of the iron oxide nanoparticle” should be understood to mean asurface of the coating portion, a surface of the iron oxide portion, orboth. In some embodiments, the extract phytochemicals are attached to,disposed upon, acting as a surface ligand for, or otherwise interactingwith the coating portion of the iron oxide nanoparticle. In someembodiments, the extract phytochemicals are attached to, disposed upon,acting as a surface ligand for, or otherwise interacting with the ironoxide portion of the iron oxide nanoparticle. In such embodiments, thecoating should not prevent the extract phytochemicals from directinteraction with the iron oxide portion of the iron oxide nanoparticle.In some embodiments, the coating is porous, the pores allowing fordirect interaction of the extract phytochemicals and the iron oxideportion. Alternatively, the coating may be attached to, disposed upon,encapsulating, or otherwise interacting with the extract phytochemicals,which are themselves in direct contact with the iron oxide portion. Suchembodiments may be thought of as sandwiching the extract phytochemicalsbetween the iron oxide portion and the coating portion. Examples ofmaterials which may comprise the coating include, but are not limited tosilica, lipids, polymers, and carbon nanomaterials. In general, thepolymer may be any suitable polymer known to one or ordinary skill inthe art. Examples of such suitable polymer include, but are not limitedto polycarboxylic acid polymers and copolymers including polyacrylicacids; acetal polymers and copolymers; acrylate and methacrylatepolymers and copolymers (e.g., n-butyl methacrylate); cellulosicpolymers and copolymers, including cellulose acetates, cellulosenitrates, cellulose propionates, cellulose acetate butyrates,cellophanes, rayons, rayon triacetates, and cellulose ethers such ascarboxymethyl celluloses and hydoxyalkyl celluloses; polyoxymethylenepolymers and copolymers; polyimide polymers and copolymers such aspolyether block imides, polyamidimides, polyesterimides, andpolyetherimides; polysulfone polymers and copolymers includingpolyarylsulfones and polyethersulfones; polyamide polymers andcopolymers including nylon 6,6, nylon 12, polycaprolactams andpolyacrylamides; resins including alkyd resins, phenolic resins, urearesins, melamine resins, epoxy resins, allyl resins and epoxide resins;polycarbonates; polyacrylonitriles; polyvinylpyrrolidones (crosslinkedand otherwise); polymers and copolymers of vinyl monomers includingpolyvinyl alcohols, polyvinyl halides such as polyvinyl chlorides,ethylene-vinylacetate copolymers (EVA), polyvinylidene chlorides,polyvinyl ethers such as polyvinyl methyl ethers, polystyrenes,styrene-maleic anhydride copolymers, styrene-butadiene copolymers,styrene-ethylene-butylene copolymers (e.g., apolystyrenepolyethylene/butylene-polystyrene (SEBS) copolymer, availableas Kraton.®. G series polymers), styrene-isoprene copolymers (e.g.,polystyrene-polyisoprene-polystyrene), acrylonitrile-styrene copolymers,acrylonitrile-butadiene-styrene copolymers, styrene-butadiene copolymersand styrene-isobutylene copolymers (e.g., polyisobutylene-polystyreneblock copolymers such as SIBS), polyvinyl ketones, polyvinylcarbazoles,and polyvinyl esters such as polyvinyl acetates; polybenzimidazoles;ionomers; polyalkyl oxide polymers and copolymers including polyethyleneoxides (PEO); glycosaminoglycans; polyesters including polyethyleneterephthalates and aliphatic polyesters such as polymers and copolymersoflactide (which includes lactic acid as well as d-, I- and mesalactide), epsilon-caprolactone, glycolide (including glycolic acid),hydroxybutyrate, hydroxyvalerate, para-dioxanone, trimethylene carbonate(and its alkyl derivatives), 1,4-dioxepan-2-one, 1,5-dioxepan-2-one, and6,6-dimethyl-1,4-dioxan-2-one (a copolymer of polylactic acid andpolycaprolactone is one specific example); polyether polymers andcopolymers including polyarylethers such as polyphenylene ethers,polyether ketones, polyether ether ketones; polyphenylene sulfides;polyisocyanates; polyolefin polymers and copolymers, includingpolyalkylenes such as polypropylenes, polyethylenes (low and highdensity, low and high molecular weight), polybutylenes (such aspolybut-1-ene and polyisobutylene), poly-4-methyl-pen-1-enes,ethylene-alpha-olefin copolymers, ethylene-methyl methacrylatecopolymers and ethylene-vinyl acetate copolymers; polyolefin elastomers(e.g., santoprene), ethylene propylene diene monomer (EPDM) rubbers,fluorinated polymers and copolymers, including polytetrafluoroethylenes(PTFE), poly(tetrafluoroethylene-co-hexafluoropropene) (FEP), modifiedethylene-tetrafluoroethylene copolymers (ETFE), and polyvinylidenefluorides (PVDF); silicone polymers and copolymers; polyurethanes;p-xylylene polymers; polyiminocarbonates; copoly(ether-esters) such aspolyethylene oxide-polylactic acid copolymers; polyphosphazines;polyalkylene oxalates; polyoxaamides and polyoxaesters (including thosecontaining amines and/or amido groups); polyorthoesters; biopolymers,such as polypeptides, proteins, polysaccharides and fatty acids (andesters thereof), including fibrin, fibrinogen, collagen, elastin,chitosan, gelatin, starch, glycosaminoglycans such as hyaluronic acid;as well as blends and further copolymers of the above. The coating maybe intended to be broken down, degraded, disintegrated, or otherwiseremoved from the iron oxide nanoparticle in whole or in part. Suchremoval may cause or coincide with release of the extract phytochemicalsfrom the iron oxide nanoparticle. In preferred embodiments, the ironoxide nanoparticles are substantially free of a coating.

The present disclosure also relates to a method of killing or inhibitingthe growth of bacteria and/or fungus, the method comprising exposing thebacteria and/or fungus to the iron oxide nanoparticles. In general, theexposing may be accomplished by any suitable type of exposing known toone of ordinary skill in the art. In some embodiments, the iron oxidenanoparticles may be used as a component in an antibacterialcomposition. In general, the antibacterial composition may take anysuitable form known to one of ordinary skill in the art. Examples ofsuch forms include, but are not limited to a solid, liquid, gel, foam,dispersion, colloid, or other type of mixture. In some embodiments, thenanoparticles are homogenously distributed throughout the volume of themixture. In some embodiments, the nanoparticles are non-homogenouslydistributed throughout the volume of the mixture. In some embodiments,the nanoparticles may separate from other components of the mixture andrequire mixing or redispersion before use.

In some embodiments, the antibacterial composition is intended for usein conjunction with exposure to visible wavelengths of light. In someembodiments, the antibacterial composition has a mode of action thatresults from the photocatalytic properties of the nanoparticles. In someembodiments, the antibacterial composition is dissolvable or dispersiblein water and may form a component of a water purification composition.When used as a component of such a water purification composition, thenanoparticles may be removed from the water or left in the water. Insuch an application, the nanoparticles may, in addition to acting in theantibacterial composition, also act in another composition such as onethat removes other substances from water that may be undesirable.

Iron oxides (red, yellow, and black) are currently approved as “exemptfrom certification” as direct food additives and are “GenerallyRecognized as Safe” as indirect food additives by the US FDA and areapproved for use as a food additive in the European Union (E172). Theantibacterial composition comprising the nanoparticles may find use as afood additive. In some embodiments, the nanoparticles may be addeddirectly to a foodstuff to form an antibacterial composition thatcomprises the nanoparticles and the components of the foodstuff. In someembodiments, the antibacterial composition is pre-formed from othercomponents before being added to the foodstuff.

Iron oxide is currently a common component in many cosmetics and bathproducts. The antibacterial composition may also find use in suchproducts. In some embodiments, the antibacterial composition comprisingthe nanoparticles is such a cosmetic or bath product. In someembodiments, the antibacterial composition is a component of a cosmeticor bath product that shows antibacterial activity. Examples of suchcosmetics or bath products include but are not limited to soaps, facialsoaps, facial washes, body washes, shampoos, conditioners, deodorants,antiperspirants, combination deodorants/antiperspirants, fragrances,foot powders, hair dyes or colors, makeup, nail products, personalcleanliness products, shaving products, depilatories, skincare products,tanning products, body or face creams, moisturizers, and anti-acneproducts.

In some embodiments, the antibacterial composition is not intended forbodily contact or ingestion. In some embodiments, the antibacterialcomposition is intended to be used in a container, pipe, reservoir, orother such vessel intended to store or transport material, or on asurface. In some embodiments the antibacterial composition is designedto be transiently contacted with the vessel or surface and then removed.In some embodiments, the antibacterial composition is designed to be incontact with the vessel or surface for an extended period of timeincluding the lifetime of either the antibacterial composition or thevessel or surface. In some embodiments, the vessel or surface may allowthe nanoparticles to be illuminated by visible wavelengths of light.

In some embodiments, the antibacterial composition further comprises asurfactant. A surfactant may be present at a weight percentage in arange of 0.02-10 wt %, preferably 0.1-5 wt %, more preferably 0.5-2 wt%. Examples of surfactants and surfactants types that may be included inthe antibacterial composition may be those surfactants/surfactant typesdescribed previously.

In one embodiment, the antibacterial composition may further comprise amutual solvent. A mutual solvent may be present at a weight percentageof 1-20 wt %, preferably 3-15 wt %, more preferably 4-12 wt %. Asdefined herein, a “mutual solvent” is a liquid that is substantiallysoluble in both aqueous and oleaginous fluids, and may also be solublein other well treatment fluids. As defined here, “substantially soluble”means soluble by more than 10 grams mutual solvent per liter fluid,preferably more than 100 grams per liter. Mutual solvents are routinelyused in a range of applications, controlling the wettability of contactsurfaces before and preventing or stabilizing emulsions.

Examples of the mutual solvent include propylene glycol, ethyleneglycol, diethylene glycol, glycerol, and 2-butoxyethanol. In a preferredembodiment, the mutual solvent is 2-butoxyethanol, which is also knownas ethylene glycol butyl ether (EGBE) or ethylene glycol monobutyl ether(EGMBE). In alternative embodiments, the mutual solvent may be one oflower alcohols such as methanol, ethanol, 1-propanol, 2-propanol,n-butanol, n-hexanol, 2-ethylhexanol, and the like, other glycols suchas dipropylene glycol, polyethylene glycol, polypropylene glycol,polyethylene glycol-polyethylene glycol block copolymers, and the like,and glycol ethers such as 2-methoxyethanol, diethylene glycol monomethylether, and the like, substantially water/oil-soluble esters, such as oneor more C2-esters through C10-esters, and substantiallywater/oil-soluble ketones, such as one or more C2-C10 ketones.

In some embodiments, the antibacterial composition may further comprisea buffer. As used herein, a buffer (more precisely, pH buffer orhydrogen ion buffer) refers to a mixture of a weak acid and itsconjugate base, or vice versa. Its pH changes very little when a smallor moderate amount of strong acid or base is added to it and thus it isused to prevent changes in the pH of a solution. Buffer solutions areused as a means of keeping pH at a nearly constant value in a widevariety of chemical applications. Examples of buffers include, but arenot limited to, HEPES buffer, TAPS, Bicine, Glycylglycine, Tris, HEPPSO,EPPS, HEPPS, POPSO, N-ethylmorpholine, TEA (Triethanolamine), Tricine,TAPSO, DIPSO, TES, BES, phosphoric acid, MOPS, imidazole PIPES and thelike.

In one embodiment, the antibacterial composition may further compriseother components, such as alcohols, glycols, organic solvents,fragrances, dyes, dispersants, non-buffer pH control additives, acids orbases, water softeners, bleaching agents, foaming agents, antifoamingagents, catalysts, corrosion inhibitors, corrosion inhibitorintensifiers, viscosifiers, diverting agents, oxygen scavengers, carrierfluids, fluid loss control additives, friction reducers, stabilizers,rheology modifiers, gelling agents, scale inhibitors, breakers, salts,crosslinkers, salt substitutes, relative permeability modifiers, sulfidescavengers, fibers, microparticles, bridging agents, shale stabilizingagents (such as ammonium chloride, tetramethyl ammonium chloride, orcationic polymers), clay treating additives, polyelectrolytes,non-emulsifiers, freezing point depressants, iron-reducing agents, otherbiocides/bactericides and the like, provided that they do not interferewith the antibacterial activity of the nanoparticles as describedherein.

In some embodiments, the bacteria and/or fungus is in the form of abiofilm.

In some embodiments, the bacteria is a gram-positive bacteria. In someembodiments, the bacteria is a gram-negative bacteria. In someembodiments, the bacteria is P. aeruginosa. In some embodiments, thebacteria is S. aureus. In some embodiments, the fungus is C. albicans.In some embodiments, the bacteria and/or fungus is at least one selectedfrom the group consisting of P. aeruginosa, S. aureus, and C. albicans.

In some embodiments, the iron oxide nanoparticles have a minimuminhibitory concentration (MIC) for P. aeruginosa of 0.60 to 1.5 mg ironoxide nanoparticles per mL, preferably 0.68 to 1.40 mg, preferably 0.75to 1.35 mg, preferably 0.9 to 1.2 mg, preferably 1.0 to 1.1 mg ironoxide nanoparticles per mL. In some embodiments, the iron oxidenanoparticles have a MIC for S. aureus of 0.9 to 2.45 mg, preferably0.95 to 2.39 mg, preferably 1 to 2.3 mg, preferably 1.25 to 2.15 mg,preferably 1.3 to 2.1, preferably 1.4 to 2.0 mg preferably 1.5 to 1.9mg, preferably 1.6 to 1.75 mg, preferably 1.65 to 1.70 mg iron oxidenanoparticles per mL. In some embodiments, the iron oxide nanoparticleshave a MIC for C. albicans of 1.30 to 2.85 mg, preferably 1.36 to 2.80mg, preferably 1.4 to 2.75 mg, preferably 1.5 to 2.6 mg, preferably 1.6to 2.5 mg, preferably 1.75 to 2.3 mg, preferably 1.9 to 2.2 mg,preferably 2.0 to 2.1 mg iron oxide nanoparticles per mL.

The present disclosure also relates to a method of treating coloncancer, the method comprising administering to a patient in need oftherapy an effective dose of the iron oxide nanoparticles. In general,the administering may be performed by any route known to one of ordinaryskill in the art.

In some embodiments, the iron oxide nanoparticles are administered as apharmaceutical composition comprising the iron oxide nanoparticles. Insome embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions. Modifications can be made to the iron oxide nanoparticlesof the present invention to affect solubility or clearance of thecompound, for example additional surface ligands and/or coatings.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, rectal administration, and direct injection into theaffected area, such as direct injection into the digestive system orcolon. Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof The proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the ironoxide nanoparticles in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation arevacuum drying and freeze-drying that yields a powder of the iron oxidenanoparticles plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. The oral compositions can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the iron oxide nanoparticles can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier, wherein theiron oxide nanoparticles in the fluid carrier is applied orally andswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the iron oxide nanoparticles aredelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the iron oxide nanoparticles are formulatedinto ointments, salves, gels, or creams as generally known in the art.

The iron oxide nanoparticles can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the iron oxide nanoparticles are prepared withcarriers that will protect the iron oxide nanoparticles against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals. Dosage unit forms include solid dosage forms,like tablets, powders, capsules, suppositories, sachets, troches andlosenges as well as liquid suspensions and elixirs. Capsule dosages, ofcourse, will contain the iron oxide nanoparticles within a capsule whichmay be made of gelatin or other conventional encapsulating material.Tablets and powders may be coated. Tablets and powders may be coatedwith an enteric coating. The enteric coated powder forms may havecoatings comprising phthalic acid cellulose acetate,hydroxypropylmethyl-cellulose phthalate, polyvinyl alcohol phthalate,carboxymethylethylcellulose, a copolymer of styrene and maleic acid, acopolymer of methacrylic acid and methyl methacrylate, and likematerials, and if desired, they may be employed with suitableplasticizers and/or extending agents. A coated tablet may have a coatingon the surface of the tablet or may be a tablet comprising a powder orgranules with an enteric-coating.

The dosage forms include dosage forms suitable for oral, buccal, rectal,parenteral (including subcutaneous, intramuscular, and intravenous),inhalant and ophthalmic administration. Although the most suitable routein any given case will depend on the nature and severity of thecondition being treated, the most preferred route of the presentinvention is oral. The iron oxide nanoparticles may be convenientlypresented in unit dosage form and prepared by any of the methodswell-known in the art of pharmacy.

In some embodiments, the iron oxide nanoparticles are administered in anamount sufficient to provide a concentration of 15 to 200 μg, preferably25 to 175 μg, preferably 50 to 150 μg iron oxide nanoparticles per mL oftumor volume at a colon cancer-containing site.

The examples below are intended to further illustrate protocols for andare not intended to limit the scope of the claims.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

EXAMPLES Preparations of Aqueous Liv52 Extract (L52E)

Liv52 tablets (Himalaya Herbals, India) were purchased and were crushedin fine powder form using mortar and pestle. About 10 g of fine powderwas then suspended in 100 ml of sterile water overnight. The suspensionswere then centrifuged and supernatant was collected. The supernatant wasfurther passed through Whatman No. 1 filter paper and finally filtratewas collected and stored at 4° C.

Liv52 Drug Inspired Synthesis of γ-Fe₂O₃ Nanoparticles

In a typical reaction procedure, 30 ml of aqueous solution of Liv52extracts was added to 70 ml of 1mM aqueous solutions of ferric chloridehexahydrate (FeCl₃·6H₂O) for the synthesis of γ-Fe₂O₃ NPs. The reactionmixture was stirred for 60 min at 65° C. Change in color of the mixtureindicates the formation of γ-Fe₂O₃ NPs. The obtained suspensions werecentrifuged at 10000 rpm and then washed with sterile water and ethanolseveral times to remove the impurities and finally dried under vacuum toobtain the γ-Fe₂O₃ NPs in powder form.

UV-Vis Spectroscopy and FTIR Analysis

Formation of γ-Fe₂O₃ NPs was carried out and confirmed by using UV-Visspectroscopy as previously described [Ashraf J M, et. al., MolecularNeurobiology. 2018; 55(9):7438-52]. FTIR analysis was employed in thespectral region of 400-4000 cm⁻¹ to recognize the presence of functionalgroups in the aqueous extract of polyherbal Liv52 drug that take part inthe synthesis and capping of NPs.

Electron Microscopic and EDS Analysis of γ-Fe₂O₃ NPs

Morphological features of the synthesized γ-Fe₂O₃ NPs was investigatedby using SEM and TEM as protocol described elsewhere [Ansari M A, et.al., Biomolecules. 2020; 10(2):336]. The γ-Fe2O3 NPs were sonicated for10 min before being used. Further, the elemental composition ofLiv52-mediated bioinspired γ-Fe₂O₃ NPs was carried out by using energydispersive spectroscopy (EDS; JED-2300 Japan).

XRD Analysis of γ-Fe₂O₃ NPs

The crystalline structure and particle size of the powdered NPs wasdetermined using an XRD machine as described in Anasari, et. al. [AnsariMA, et. al., Biomolecules. 2020; 10(2):336].

Vibrating-Sample Magnetometer (VSM) Analysis of γ-Fe₂O₃ NPs

The measurement of magnetic properties of the synthesized γ-Fe₂O₃ NPswas performed using a vibrating sample magnetometer at room temperature.

GC-MS Analysis of Liv52 Extract

The investigation of presence of bioactive compounds in methonolicextract of Liv52 was analyzed by GC-MS as protocol described by Ali etal. [Ali S G, et. al., Journal of basic microbiology. 2017;57(3):193-203].

Antibacterial, Anticandidal and Anti-Biofilm Studies of γ-Fe₂O₃ NPs

Biofilm-producing strains of multi drug resistant Pseudomonas aeruginosa(MDR-PA), Methicillin-resistant Staphylococcus aureus (MRSA) and Candidaalbicans were used for antibacterial, anticandidal and antibiofilmstudy.

Determination of Minimal Inhibitory Concentration (MIC)

Microbroth dilution method was used to determine the MIC of γ-Fe₂O₃ NPsas described by Balasamy, et. al. [Balasamy R J, et. al., RSC Advances.2019; 9(72):42395-408]. The bacterial and Candida strains treated withtwo-fold serial dilutions of γ-Fe₂O₃ NPs (0.156-10 mg/ml) were incubatedat 37° C. and 28° C., respectively for overnight. The MIC was defined asthe lowest concentration of tested NPs at which no visible growth of thetested bacteria and Candida was observed.

Minimal Bactericidal and Fungicidal Concentration (MBC/MFC)

After MIC assessment of γ-Fe₂O₃ NPs, aliquots of 100 μl from wells inwhich no visible bacterial and fungal growth was seen were furtherinoculated on MHA and SDA plates for 24 h at 37° C. and 28° C.,respectively. The MBC/MFC endpoint is defined as the lowestconcentration of tested NPs that kills 100% population of testedbacterial and Candidal strains.

Effect of γ-Fe₂O₃ on Biofilm Forming Abilities of MRSA, MDR-PA and C.albicans

The inhibition of biofilm formation after treatment with γ-Fe₂O₃ NPs wasquantitatively examined by microtiter crystal violet assay as describedby Balasmy, et. al. [Balasamy R J, et. al., RSC Advances. 2019;9(72):42395-408]. Briefly, fresh cultures of 20 μl of bacteria and yeast(C. albicans) were inoculated in 180 μl of different concentrations ofsynthesized γ-Fe₂O₃ NPs (0.3125-2.5 mg/ml) and then bacteria wereincubated at 37° C. and yeast at 28° C., respectively. MRSA, MDR-PA andC. albicans without NPs were considered as control group. Afterovernight of incubation, the content from the microtiter wells weredecanted and gently washed with 1× PBS thrice using ELISA washer andleft the microtiter plate for drying. The adhered biofilms was thenstained with crystal violet solution (0.1% w/v) for 15 min. Afterstaining, the overflow dyes were decanted and washed again with PBS anddried the wells completely. After drying, the stained biofilm wassolubilised with 95% ethyl alcohol and then optical density was taken at595 nm using ELISA reader.

Visualization of Biofilm Architecture: SEM Analysis

The effect of γ-Fe₂O₃ NPs on MRSA, MDR-PA and C. albicans biofilmarchitecture was investigated by SEM as described by Jalal, et. al.[Jalal M, Artificial cells, nanomedicine, and biotechnology. 2018;46(sup1):912-25]. In brief, 100 μl fresh cultures of tested bacterialand yeast strains with and without L52E-γ-Fe₂O₃ NPs were inoculated onglass coverslips in a 12-wells plate for overnight at 37° C. and 28° C.,respectively. After incubation, the glass coverslips were taken off andwashed with 1× PBS to remove the unadhered cells. After washing, thecoverslips were primarily fixed with glutaraldehyde (2.5% v/v) for 24 hat 4° C. After fixation, washed the coverslips again and then subjectedit to dehydration (a series of ethyl alcohol) and drying. After drying,gold coating of treated and untreated samples were performed and thenthe effects of γ-Fe₂O₃ NPs on biofilms of tested bacteria and yeast wereobserved using SEM.

Ultrastructural Alteration in Bacterial and Candida Cells Caused by NPs

The ultrastructural changes caused by L52E-γ-Fe₂O₃ NPs in testedbacterial and yeast strains cells were examined by SEM as protocoldescribed by Shukla et al. [Shukla A K, et. al., Materials Chemistry andPhysics. 2019; 233:102-12]. Briefly, ˜10⁶ CFU/ml of MRSA, MDR-PA and C.albicans were inoculated in a 2 ml sterile tubes with and withoutγ-Fe₂O₃ NPs and then incubated at 24 h at required temperature. Afterincubation, cells were washed three to four times and fixed with primaryfixative glutaraldehyde (4% v/v). After primary fixation, cells wereagain fixed with secondary fixative i.e., 1% osmium tetroxide for 1 hand then subjected to dehydration (a series of ethanol), drying and goldcoating, and then finally observed the effects of NPs on morphology oftested bacteria and Candida using SEM at an accelerated voltage of 20EV.

Evaluation of Anticancer Potential of γ-Fe₂O₃ NPs

Human colorectal carcinoma cell line (HCT-116 cells) was used toevaluate the anticancer potential of γ-Fe₂O₃ NPs. 96-well cell cultureplates were used for drug treatments using the procedure described byKhan et al. [Khan F A, et. al., Artificial Cells, Nanomedicine, andBiotechnology. 2018; 46(sup3):S247-53]. The cancer cells were treatedwith different concentrations of γ-Fe₂O₃ NPs. All the treatments wereperformed in triplicate for statistical calculations.

MTT Assay

The MTT assay was used to assess and measure the cell metabolic activityand cytotoxicity of γ-Fe₂O₃ NPs. MTT assay was performed in 96 wellculture plates by measuring optical density at 570 nm and cell viability(%) was using equation (1):

$\begin{matrix}{{\%{of}{cell}{viability}} = {\frac{{{Optical}{density}{of}{nanoparticles}} - {{treated}{cells}}}{{Optical}{density}{of}{control}{cells}} \times 100}} & (1)\end{matrix}$

Cell Morphology

The effects of different concentrations of γ-Fe₂O₃ NPs on the anatomyand morphology of Human colorectal carcinoma cell line was analyzed atthe end of experiments under an inverted microscope equipped with adigital camera.

Biosynthesis and UV-Vis Analysis of γ-Fe₂O₃ NPs

Aqueous extract of Liv52, a traditional polyherbal drug was used as areducing, stabilizing, and capping agent for the synthesis of γ-Fe₂O₃NPs. The color of the FeCl₃·6H₂O after addition of extract change fromcolourless to dark brown to black precipitates indicated the formationof iron oxide nanoparticles (IONPs). The biosynthesis of γ-Fe₂O₃ NPs wasconfirmed by UV-Vis absorbance spectroscopy which showed a maximumabsorbance at 327 nm due to surface plasmon resonance (FIG. 1 ) and issupported by the results of Balamurughan et al. [Balamurughan M G, et.al., Journal of Chemical and Pharmaceutical Sciences. 2014; 4:201-204].It has been reported that IONPs shows absorbance peak between 200 to 400nm [Klačanová K, et. al., Journal of Chemistry. 2013, Article ID 961629;and Suganya D, et. al., Int J Curr Res. 2016; 8(10:42081-5].

FTIR and GC-MS Analysis

FTIR analysis were performed to identify the possible phytocompoundspresent in the Liv52 extract that were responsible for the reduction ofFe³⁺ ions and capping as well as stabilization of the reduced iron oxidenanoparticles. The FTIR spectrum of Liv52 extract (FIG. 2 ) representsthe major absorption spectra at 3234 cm⁻¹ corresponding to hydroxylgroup of polyphenolic compounds, 1633 cm⁻¹ corresponds amide groupwhereas peak 2135 cm⁻¹ corresponds to the N—H/C—O stretching vibration(see Balamurughan et al.). The FTIR spectrum of green synthesized IONPs(FIG. 2 ) represents the shift in peak from 3234 to 3267 cm⁻¹, 2135 to2146 cm⁻¹ and 1633 to 1645 cm⁻¹ in comparison with the FTIR spectrum ofL52E. The peak 638 cm⁻¹ indicated the Fe—O vibration of Fe₂O₃nanoparticles, as reported previously which indicated the formation ofIONPs [Gotić M, et. al., Materials Research Bulletin. 2009;44(10):2014-21]. The peak at 1645 cm¹ indicate the presence of carbonylgroups (C═O) of long chain carboxylic fatty acids and polyphenoliccompounds present in L52E that might be responsible for the reduction ofFe ions to iron oxide NPs.

The GC-MS analysis of L52E was performed to identify and confirm thephytochemicals present in L52E that are responsible for reduction of Feions and capping and stabilization of nanoparticles. The GS-MS of L52Eshows 37 peaks (FIG. 3A). The major bioactive phytochemicals present inL52E are; n-Hexadecanoic acid (21.95%), 9,12-Octadecadienoic acid(Z,Z)-(20.45%), 9-Octadecenoic acid (Z)-(18.01%), Octadecanoic acid(13.99%), (Z)-3-(pentadec-8-en-1-yl)phenol (11.92%), Piperine (2.22%),1,3-Propanediol, 2-(hydroxymethyl)-2-nitro-(1.30%) and Tetradecanoicacid (1.14%) on to surface of γ-Fe₂O₃NPs could be argued as mainbio-fabricating and stabilizing agents (Table. 1; FIG. 3B). It has beensuggested that some of these bioorganic moieties of L52E possiblyinteract with Fe³⁺ ions during the reduction and nucleation process,which ultimately results the formation of nanoparticles [Makarov V V,et. al., Langmuir. 30 (2014) 5982-5988]. It has been suggested that theabsorption of L52E on nanoparticle surface was due to ionic interaction,hydrogen bonding, π-π interactions, or reversible bond formation, whichprovide stability to the bio-fabricated nanoparticles [Silva A B, et.al., Corr. Sci. 48 (2006) 3668-3674; Olivares O, et. al., Appl. Surf.Sci. 252 (2006) 2894-2909; and Bereket G,. Yurt A., Con. Sci. 43 (2001)1179-1195]. In addition, it has been observed that some phytocompoundspresent in L52E contains N, O, Si and P atoms, may also form coatings onmetal nanoparticles to protect the nanoparticles from oxidation andcorrosion [James A O, Atela A O, Int. J. Pur. Appl. Chem. 3 (2008)159-163; and Abiola O K,. James A O, Corros. Sci. 52 (2010) 661-624].Recently, Ali et al. have also demonstrated adsorption of organicmolecules such as oxime-, methoxy-phenyl (C₈H₉NO₂), hexadecanoic acid(C₁₆H₃₂O₂), cyclohexanol, 2,6-dimethyl (C₈H₁₆O) and ethanone,1-phenyl(C₈H₈) on the surface of hematite (α-Fe₂O₃) nanoparticles synthesized byAloe vera extract [Ali K, et. al., Journal of Photochemistry andPhotobiology B: Biology. 2018; 188:146-58]. Full results are presentedin Table 1 below.

TABLE 1 GC-MS analysis of phytocomponents present in Liv52 extract PeakRetention area Mol. Mol. Peak Time (%) Name of compounds weight formulaCAS Class  1 14.796 0.16 Octanoic Acid, 3-Oxo-, Methyl C₉H₁₆O₃ 17222348- Methyl ester   Ester 95-4  2 15.750 1.30 1,3-Propanediol, 2-C₄H₉NO₅ 151 126- Isobutyl   (hydroxymethyl)-2-nitro- 11-4 Glycerol,  nitro/alkaloid  3 16.145 0.11 Benzene, 1-(1,5-Dimethyl-4- C₁₅H₂₂ 202644- aromatic   Hexenyl)-4-Methyl- 30-4 curcumene  4 17.794 0.171-Tridecanol C₁₃H₂₈O 200 112- Aliphatic   70-9 alcohol  5 18.886 0.86Ar-tumerone C₁₅H₂₀O 216 532- aromatic   65-0 compound   ketone  6 19.1550.55 Mome Inositol C₇H₁₄O₆ 194 0-0-0 carbocyclic   sugar  7 19.441 0.25Alpha.- tumerone C₁₅H₂₂O 218 82508- aliphatic   15-4 ketone  8 20.1651.14 Tetradecanoic acid C₁₄H₂₈O₂ 228 544- Fatty acid   63-8  9 20.6040.10 1-Tetradecanol C₁₄H₃₀O 214 112- saturated fatty 72-1 alcohol 1021.174 0.10 Neophytadiene C₂₀H₃₈ 278 504- Alkene 96-1 compound 11 21.4890.19 Didodecyl phthalate C₃₂H₅₄O₄ 502 2432- Aromatic 90-8 carboxylicacid 12 22.093 0.09 7,9-Di-tert-butyl-1- C₁₇H₂₄O₃ 276 82304 Ketoneoxaspiro(4,5)deca-6,9-diene-2,8- 66-3 compound dione 13 22.298 0.25Hexadecanoic acid, Methyl Ester C₁₇H₃₄O₂ 270 112- fatty acid 39-0 methylesters. 14 22.493 0.33 9-Eicosyne C₂₀H₃₈ 278 71899- Aliphatic 38-2alkyne 15 22.790 21.95 n-Hexadecanoic acid (Palmitic acid) C₁₆H₃₂O₂ 25657-10-3 saturated fatty acid 16 23.267 0.25Azuleno[4,5-b]furan-2(3H)-one, C₁₅H₁₈O₂ 230 477- Ketone 43-0 compounds17 23.726 0.25 Palmitic Acid, TMS derivative C₁₉H₄₀O₂Si 328 55520- Fattyacid 89-3 silyl ester 18 24.325 0.32 9,12-Octadecadienoic acid, methylC₁₉H₃₄O₂ 294 2462- Ester ester 85-3 19 24.822 20.45 9,12-Octadecadienoicacid (Z,Z)- C₁₈H₃₂O₂ 280 60-33-3 polyunsaturated (Linoleic Acid) omega-6fatty acid 20 24.887 18.01 9-Octadecenoic acid (Z)-(Oleic C₁₈H₃₄O₂ 282112- monounsaturated acid) 80-1 omega-9 fatty acid, 21 25.147 13.99Octadecanoic acid (Stearic acid) C₁₈H₃₆O₂ 284 57-11-4 saturated fattyacid 22 26.608 0.45 1-(4-Hydroxy-3- C₁₇H₂₄O₃ 276 555- Aromaticmethoxyphenyl)dec-4-en-3-one 66-8 Phenolic 23 27.083 0.73Z,Z-8,10-Hexadecadien-1-ol C₁₆H₃₀O 238 14724 Aliphatic 0-92-4 alcohol 2427.323 0.42 9-Octadecenoic acid (Z)- C₁₈H₃₄O₂ 282 112- monounsaturated80-1 omega-9 fatty acid, 25 27.693 0.29 Phosphonic acid, dioctadecylester C₃₆H₇₅O₃P 586 19047- ester 85-9 26 28.658 0.42 Ginkgol (TMS)C₂₄H₄₂OSi 374 0-00-0 Aromatic phenols 27 28.811 11.92(Z)-3-(pentadec-8-en-1-yl)phenol C₂₁H₃₄O 302 501- Aromatic (Cardanolmonoene) 26-8 phenols 28 29.102 0.17 Phenol, 3-pentadecyl- C₂₁H₃₆O 304501- Aromatic 24-6 phenols 29 33.378 0.39 Piperine C₁₇H₁₉NO₃ 285 94-622alkaloid 30 34.058 0.26 Squalene C₃₀H₅₀ 410 111- Triterpenes 02-4 3135.088 1.83 Piperine C₁₇H₁₉NO₃ 285 94-62- alkaloid 2 32 35.594 0.16(2E,4E,10E)-N-Isobutylhexadeca- C₂₀H₃₅NO 305 94354 alkaloid2,4,10-trienamide 6-13-2 33 36.190 0.66 Doconexent, TBDMS derivativeC₂₈H₄₆O₂Si 442 20871 Fatty acid 4-15-2 silyl ester 34 38.547 0.34Stigmasta-5,22-dien-3-ol, C₂₉H₄₈O 412 83-48- steroid (3.beta.,22E)- 7alcohols 35 39.357 0.72 Stigmast-5-en-3-ol, (3.beta.)- C₂₉H₅₀O 41483-46- steroid 5 alcohols 36 40.237 0.19 2-Hydroxymethyl-2,6,8,8-C₁₆H₂₈O 236 13723 alcohol tetramethyltricyclo[5.2.2.0(1,6)] 5-47-3undecane 37 40.696 0.25 Lupeol C₃₀H₅₀O 426 545-47-1 triterpenoid 100Electron Microscopic Properties of L52E-γ-Fe₂O₃ NPs

The SEM micrographs revealed that the synthesized nanoparticles weremostly agglomerated and were irregular and spongy in appearance withrough surfaces (FIG. 4A). Further, to confirm the average size, shapeand structural morphology of the as-synthesized nanoparticles, thesamples were analyzed by TEM. The TEM micrographs clearly shows that thesynthesized nanoparticles were well separated, almost uniform indistribution and roughly spherical in shape (FIG. 4B). Particledistribution was analysed by ImageJ software and it was found that theaverage particles size of the synthesized γ-Fe₂O₃ NPs was 30.66 nm (FIG.4D). In this study, the size of the as-synthesized NPs was found inagreement to the size calculated by XRD analysis (28.52 nm). Further,the elemental composition of synthesized γ-Fe₂O₃ NPs was determined byusing EDX (FIG. 4C) which shows prominent peak of oxygen (33.95%),chloride (21.45%), silicon (2.87%) and potassium (1.5%) along with threecharacteristic peaks of Fe (40.23%) at approximate 0.5, 6.5 and 7.0 keV,respectively (FIG. 4C).

XRD Analysis of γ-Fe₂O₃ NPs

The structure and crystallite phase of biosynthesized magneticnanoparticles annealed at 60° C. was obtained by X-ray diffraction (XRD)in the 20 range from 20-80° (FIG. 5 ). It was found that the peaks ofas-prepared nanoparticles are in good agreement with the reference datafor the JCPDS file (39-1346), and can be indexed to the cubic spinelstructure and were identified as maghemite (γ-Fe₂O₃) [Wu W, et. al.,Science and Technology of Advanced Materials. 2015; 16: 023501 (43 pp),doi:10.1088/1468-6996/16/2/023501; and. Wu W, et. al., Nanoscale ResLett. 2010; 5(9): 1474-1479]. The main peaks (220), (311), (440), (422),(511) and (440) were observed at 22.44°, 30.15°, 35.46°, 43.12°, 54.20,57.12° and 62.55°, respectively (FIG. 5 ), consistent with literaturevalues [Cao D, et. al., Scientific reports. 2016; 6(1):1-9]. Thepresence of characteristic peaks of γ-Fe₂O₃ at 30.15° and 43.12° assurethe formation of maghemite nanoparticles [Rana P, et. al., MaterialsScience for Energy Technologies. 2019; 2(1):15-21]. The XRD results alsoexhibits some unassigned additional peaks which might be due to thepresence of bioorganic compounds on the surface of γ-Fe₂O₃ NPs. They actas reducing and capping agent on the surface of γ-Fe₂O₃ and alsoproviding the stability to the γ-Fe₂O₃ NPs. The crystallite size (D)calculated using Scherer's formula was 28.52 nm [Ansari M A, et. al.,Biomolecules. 2020; 10(2):336].

Magnetic Properties of Biosynthesized L52E-γ-Fe₂O₃ NPs

The magnetic behaviour of biosynthesized L52E-γ-Fe₂O₃ NPs was monitoredby measuring hysteresis loop of the Fe₂O₃ NPs at room temperature (T=300K). It was found that the L52E-γ-Fe₂O₃ NPs were superparamagnetic innature at room temperature with a calculated saturation magnetization(Ms) of 21.5 emu/g (see FIG. 6 ) which is in accordance to the study ofPalanisamy et al. [Palanisamy K L, et. al., Digest Journal ofNanomaterials & Biostructures (DJNB). 2013; 8(2)].

Antibacterial and Anticandidal Activity of L52E-γ-Fe₂O₃ NPs

The antibacterial and anticandidal activity of L52E-γ-Fe₂O₃ NPs wereinvestigated against drug resistant gram-negative bacteria P.aeruginosa, gram-positive MRSA and C. albicans in a 96 well microtiterplate using microbroth dilution method. The MICs values of L52E-γ-Fe₂O₃NPs against P. aeruginosa, MRSA and C. albicans were 1.04±0.36,1.67±0.72 and 2.08±0.72 mg/ml, respectively. The MBC values for P.aeruginosa and MRSA were 2.5 and 3 mg/ml, respectively, while MFC valuewas found 5 mg/ml against C. albicans. The MIC values of L52E-γ-Fe₂O₃NPs against P. aeruginosa, MRSA and C. albicans found are consistentwith the study of Farouk et al. [Farouk F, et. al., BiotechnologyLetters. 2020; 42(2):231-40]. In a study by Behera, et. al., it has beenreported that chemically synthesized IONPs did not show any activityagainst P. aeruginosa (MTTC 1034) at 50 mg/ml [Behera S S, et. al.,World J Nano Sci Eng. 2012; 2(4):196-200]. Tran et al. reported thatIONPs completely inhibit the S. aureus growth at 3 mg/ml [Tran N, et.al., International journal of nanomedicine. 2010; 5:277]. In anotherstudy, MIC for MRSA and P. aeruginosa was 360±160 and 100±50 μg/ml,respectively [Masadeh M M, et. al., Cytotechnology. 2015; 67(3):427-35].Prodan et al. reported that IONPs did not exhibit any inhibitory effecton Candida krusei and B. subtilis growth at 5 mg/ml of concentration[Prodan A M, et. al., Journal of Nanomaterials. 201; 2013 Article ID893970]. The MIC for different species of bacterial and Candidal strainsmay differ as well because of their cell wall structures. It was foundthat gram negative bacteria were more susceptible when compared to grampositive bacteria and yeast. Previous studies also indicate thatgram-negative bacteria were more sensitive to IONPs than gram-positivebacteria [Prabhu Y T, et. al., International Nano Letters. 2015;5(2):85-92; and Salem D M, et. al., The Egyptian Journal of AquaticResearch. 2019; 45(3):197-204]. The present data suggest thatbiosynthesized γ-Fe₂O₃ NPs can be used as antimicrobial coatings ortherapeutic agents.

Interaction of L52E-γ-Fe₂O₃ NPs with Bacterial and Candidal Cells: SEMAnalysis

SEM was employed to visualize the effects of L52E-γ-Fe₂O₃ NPs on theultrastructure of P. aeruginosa (FIG. 7A-7B), MRSA (FIG. 7C-7D), and C.albicans (FIG. 7E-7F). The untreated gram-negative P. aeruginosa wereintact, normal, smooth, and typically rod shaped (FIG. 7A). However,L52E-γ-Fe₂O₃ NPs treated P. aeruginosa cells were severely damaged. Thesurface of cell envelope were abnormal in appearance and characterizedby deep depression and “pits (FIG. 7B). Further, the cells became large,swollen, and elongated that indicate the loss of membrane integrity andsevere damage of cell wall.

In case of gram-positive MRSA, it was found that control cells werenormal, intact and typically spherical in shape (FIG. 7C). However,L52E-γ-Fe₂O₃ NPs treated MRSA cells were severely damaged and werecharacterized by roughness, irregularities and depression on surface ofcell envelope (FIG. 7D). The ultrastructural alteration in gram-negativeand gram-positive bacteria as analysed by SEM shows that L52E-γ-Fe₂O₃NPs were more effective against gram-negative bacteria as compared togram-positive bacteria. This is possibly due to structural differencesin cell wall of both types of bacteria. The cell wall of gram-positivebacteria are mainly composed of a rigid and thick peptidoglycans (20-80nm) layers that give additional protection and less anchoring moietiesfor NPs. In contrast, gram-negative cell wall is composed of a thinlayer of peptidoglycan i.e., 7-8 nm [Madigan, M., Martinko, J., 2005.Brock biology of microorganisms, 11th ed. Pearson Prentice Hall, NewJersey] but highly negatively charged lipopolysaccharides layer [Salton,M. R. J., Kim, K. S., Baron, S., 1996. Medical microbiology, fourth ed.University of Texas Medical Branch, Galveston] that most likely attractsthe positively charged NPs.

The control C. albicans cells in the absence of L52E-γ-Fe₂O₃ NPsdisplayed normal morphological characteristics with a typical oval shapestructure and intact cell membrane and cell-wall (FIG. 7E). In contrast,C. albicans cells treated with L52E-γ-Fe₂O₃ were severely damaged andcells were became round and enlarged, with significant alterations incell membrane and cell-wall. Roughness and depressions on cell surfacehas been observed that indicate the loss of integrity of cell membraneand cell-wall (FIG. 7F). It is well known that the cell wall of C.albicans play an important role in adhesion and morphogeneticconversions and pathogenicity. The cell wall of C. albicans mainly iscomposed of glucans, chitin and mannoproteins that provide rigidity tothe overall cell wall structure [Chaffin W L, et. al., Mol Biol Rev.1998; 62:130-180]. FIG. 7E shows that untreated yeast cells havedistinctive cell wall and cell membrane, but cells treated withL52E-γ-Fe₂O₃ NPs shows separation of cell membrane from the cells and itwas difficult to distinguish cell wall (FIG. 7F). SEM image clearlyshows that the interaction of NPs lead to damage of the outer cell wall.These results are consistent with previous reports, where authorsreported that formation of pits and hole by NPs in yeast cell lead tothe death of C. albicans [Lara H H, et. al., Journal ofnanobiotechnology. 2015; 13(1): 1-2].

The exact mechanism of antimicrobial action of IONPs is still not clearand understood. It has been reported that the microbiocidal activity ofIONPs is due to generation of reactive oxygen species (ROS), such ashydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), superoxide radicals (O₂⁻), or hydroxyl radicals (—OH), which can damaged the proteins and DNAin the bacteria [Rudramurthy G R, et. al., Molecules. 2016; 21(7):836].Armijo et al. hypothesized that IONPs generate H₂O₂ which can penetratethe bacterial cell membrane and entered inside the intracellular spacethat results the death of bacteria [Armijo L M, et. al., Journal ofNanobiotechnology. 2020; 18(1):1-27]. Bertini et al. hypothesized thatROS generated by iron NPs may damage ferredoxins, succinatedehydrogenase, nicotinamide adenine dinucleotide dehydrogenase,hydrogenases, coenzyme Q and nitrogenise [Bertini I, et. al.,Bioinorganic chemistry. Mill Valley: University Science Books; 1994].Henle, et. al. and Touati both reported the Fenton reaction mechanismfor the antibacterial effects of iron oxide which is linked to DNAdamage and other macromolecules by production of superoxide anion(O^(.−2)) and H₂O₂ free radicals [Henle E S, Linn S. J Biol Chem. 1997;272(31):19095-8; and Touati, D., Biochem Biophys. 2000; 373(6), 1-6].Prabhu et al. also reported that ROS produced by IONPs causes theinhibition of S. aureus, E. coli, P. vulgaris and Xanthomonas [Prabhu YT, et. al., International Nano Letters. 2015; 5(2):85-92.]. Lee et al.reported that nano scale zero-valent iron NPs may penetrate the E. colimembrane and interact with intracellular oxygen and thus produceoxidative stress that ultimately interferes to bacterial cell membrane[Lee, C., et. al., Environ Sci Technol. 2008; 42(13), 4927-4933].Rezaei-Zarchi et al. reported that the antimicrobial activity of NPs waspossibly due to electromagnetic attraction between the positive chargesof NPs and the negative charges of microbe's cell wall and membranes,which oxidize and kill these microbes [Rezaei-Zarchi S, et. al., Iran.J. Pathol. 2010; 5:83-89]. However, Li, et. al. reported that the deathof bacteria by IONPs was due the penetration and interlization of NPsinside the bacterial cell that lead to formation of intracellularvacuole, swelling, rupturing and separation of the cell membrane [Li Y,et. al., Molecules. 2018; 23(3):606].

Effects of L52E-γ-Fe₂O₃ NPs on Adherence of MRSA, P. aeruginosa and C.albicans Biofilms

The antibiofilm activity of L52E-γ-Fe₂O₃ NPs against biofilm formingMRSA, multidrug-resistant P. aeruginosa, and C. albicans was assessed byits ability to disrupt biofilms formation and their adhesion grown in96-well polystyrene plate. FIG. 8 clearly shows that L52E-γ-Fe2O3 NPsinhibit biofilms formation by MRSA, P. aeruginosa and C. albicans in adose dependent manner [Shi S F, et. al., International journal ofnanomedicine. 2016; 11:6499]. At concentration of 1.25 mg/ml, theinhibition of biofilms formation by L52E-γ-Fe₂O₃ NPs was 82.3% formultidrug-resistant P. aeruginosa, 81.5% for MRSA, and 55.5% for C.albicans, respectively (FIG. 8 ). Obtained results revealed thatL52E-γ-Fe₂O₃ NPs had affinity to hinder the biofilms formation byhampering their adhesion. The present study is in accordance to study ofAli, et. al., where they reported that IONPs inhibit biofilm formationin P. aeruginosa by 84.13±6.0 at 1 mg/ml [Ali K, et. al., Journal ofPhotochemistry and Photobiology B: Biology. 2018; 188:146-58]. Taylorand Webster showed that IONPs in a concentration range of 0.01 to 2mg/mL were able to kill up to 25% of 48 h old S. epidermidis biofilm[Taylor EN, Webster T J, International journal of nanomedicine. 2009;4:145]. Prodan et al. reported that IONPs at 5 mg/mL exhibited a strongstimulatory effect on the biofilm development by P. aeruginosa 1397, E.faecalis ATCC 29212, B. subtilis, E. coli ATCC 25922, and C. krusei 963[Prodan A M, et. al., J Nanomater. 2013; 2013:587021]. In another study,it has been reported that IONPs significant reduces the biofilm growthin S. aureus and P. aeruginosa [Sathyanarayanan M B, et. al.,International Scholarly Research Notices. 2013; 2013].

Visualization of MRSA, P. aeruginosa and C. albicans Biofilms by SEM

The effect of L52E-γ-Fe₂O₃ NPs at their sub-MIC over matured biofilms,their aggregation and colonization developed on glass surface wasinvestigated by SEM (FIG. 9A-9F). SEM analysis was performed to validatethe surface morphology and anatomy of biofilms formed by testedpathogens with or without L52E-γ-Fe₂O₃ NPs. L52E-γ-Fe₂O₃ NPs treatedgroups displayed the reduction of thick aggregation of pathogenicbacteria and Candida than control (FIG. 9A-9F). This might be due to thedegradation/reduction of the thick EPS layer present in the biofilms.Thoroughly, our results have provided altogether evidences thatbiosynthesised L52E-γ-Fe₂O₃ NPs has an effective antibiofilm potentialagainst the different pathogens. The control P. aeruginosa biofilms weremuch aggregated and glass biofilm grown on coverslips surface supporthigher number of adhered cells that were mostly compact (FIG. 9A), whileL52E-γ-Fe₂O₃ NPs coated glass surfaces significantly inhibit biofilmformation and their aggregation, clumping and colonization (FIG. 9B). Itwas observed that P. aeruginosa biofilm cells had an irregular andshrivelled appearance. L52E-γ-Fe₂O₃ NPs treated group not only hamperedbiofilm formation but apparent loss of cell wall and membrane was alsoobserved on bacterial surface (FIG. 9B), indicating the severe damage ofbiofilm integrity and disruption of EPS matrix.

Similarly, uncoated control glass coverslips surface support huge numberof MRSA biofilm cells colonization, aggregation and their adherence andcells were highly arranged and clumped in chains (FIG. 9C). However,MRSA biofilms treated group shows drastically reduced, scattered andless viable cells (FIG. 9D2). Shi et al. reported that IONPs moderatelydecreased biofilm formation by S. aureus at 0.5 mg/ml, however theyreported that at 4 mg/ml, only a small protrusion of biofilm wasobserved [Shi S F, et. al., International journal of nanomedicine. 2016;11:6499]. Subbiandoss, et. al. reported that carboxyl-grafted SPIONscause ˜8-fold higher percentage of death of staphylococci biofilm thanthat of gentamicin [Subbiandoss G, et. al., Acta Biomater. 2012;8(6):2047-2055].

Biofilm-growing C. albicans cultures in the absence of L52E-γ-Fe₂O₃ NPsshowed a characteristic intense network of hyphae and highly aggregatedcells (FIG. 9E). After treatment with L52E-γ-Fe₂O₃ NPs scarce biofilmswas observed, which were composed predominantly of scattered individualC. albicans cells (FIG. 9F). Further, it has been found that the truehyphae were almost absent from these biofilms and thus L52E-γ-Fe₂O₃ NPstreatment inhibit hyphae formation and subsequently hampered biofilmformation. Treated biofilm had almost no pseudohyphae or true hyphae,and was clearly reduced in number of cells, disruption of the cell-wallis observed in treated biofilm. Similar results have been reported byLara et al. against C. albicans treated with AgNPs [Lara H H, et. al.,Journal of nanobiotechnology. 2015; 13(1):1-2]. It is important to notethat hyphae formation and development of biofilms are the two mainvirulence determinants of Candida species [Pierce C G, et. al., CurrOpin Pharmacol. 2013; 13:726-730]. Further, the mature biofilms encasedwithin extracellular matrix also make them resistant to antibiotics[Pierce C G, et. al., Nat Protoc. 2008; 3:1494-1500; and Nett J, et.al., Antimicrob Agents Chemother. 2007; 51:510-520]. The binding ofIONPs to cell membranes and/or membrane proteins may also disruptbacteria functions that may lead to bacterial cell death [Park H, et.al., J Microbiol Methods. 2011; 84(441-45].

Anticancer Properties of L52E-γ-Fe₂O₃ NPs on Colon Cancerous Cells(HCT-116)

The effects of L52E-γ-Fe₂O₃ NPs on human colorectal cancer (HCT-116)cells was evaluated by both quantitatively (MTT assay) and qualitatively(microscopic) at different concentration i.e., 10, 50 and 100 μg/ml. Thelowest dose of L52E-γ-Fe₂O₃ NPs (10 μg/ml) exhibit 99.97% cellsurvivability whereas it was found that the cells survivability wasdecreased to 36.96 and 27.08% at 50 and 100 μg/ml, respectively (FIG. 10). It has been found that L52E-γ-Fe₂O₃ NPs affects survivability ofhuman colorectal cancer cells in a dose-dependent manner. Similar resulthas been reported by Khan et al. on HCT-116 cells after treatment withfluorescent magnetic submicronic polymeric nanoparticles [Khan F A, et.al., Artificial Cells, Nanomedicine, and Biotechnology. 2018;46(sup3):S247-53]. Bai et al. reported that IONPs did not exhibit anysignificant cytotoxic effects on viability of colorectal cancer (HT29)cells at 50 μg/ml and they found that cytotoxicity was 26.98% [Bai A J,et. al., Artificial Cells, Nanomedicine, and Biotechnology. 2018;46(7):1444-51]. However, in this work, the cytotoxicity was 63.04 and72.92% at 50 and 100 μg/ml, respectively (FIG. 10 ). In another study,it has been reported that IONPs (Fe₃O₄) synthesized by peel extract ofPunica Granatum did not exhibit any effects on HCT-116 cells (IC50>250μg/ml) [Yusefi M, et. al., Journal of Molecular Structure. 202015;1204:127539].

Impact of L52E-γ-Fe₂O₃ NPs on the cell morphology of HCT-116

Further, the morphology of HCT-116 cells after 72 h post-treatments ofL52E-γ-Fe₂O₃ NPs was examined qualitatively using light microscopy. Themorphology of untreated i.e., control cells were normal and healthy andno damage has been observed (FIG. 11A). The cells treated with lowconcentration of L52E-γ-Fe₂O₃ NPs (i.e., 10 μg/mL) did not show anysignificant anatomical and morphological changes and the cell membraneand nucleus was healthy and normal (FIG. 11B). Whereas HCT-116 cellstreated at 50 μg/mL and 100 μg/mL of L52E-γ-Fe₂O₃ NPs showed significantmorphological structural changes in cell membrane and nucleus thatincludes nuclear condensation nuclear disintegration, and cell death(FIG. 11C-11D). Morphological analysis revealed that L52E-γ-Fe₂O₃ NPsproduced dose-dependent effects on HCT-116 cancer cells. It has beenreported that IONPs did not have significant toxic effect on morphologyof colorectal cancer (HT29) cells at 100 μg/mL [Bai Aswathanarayan J,et. al., Artificial Cells, Nanomedicine, and Biotechnology. 2018;46(7):1444-51]. These results are in good agreement with the previousreports which showed that niobium substituted cobalt-nickel nano-ferritecause similar cytotoxic effects on HCT-116 cells [Tombuloglu H, et. al.,Journal of Biomolecular Structure and Dynamics. 2020; 3:1-9]. Khan etal. reported that fluorescent magnetic submicronic polymericnanoparticles causes' significant nuclear condensation and fragmentationand loss of cell numbers to HCT-116 cells and they also observed thatmany dead cells and their debris were present in culture media. Though,the exact mechanism of killing of cancerous by IONPs is still unclear.It has been reported that the anticancer IONPs might be because ofbreakdown of IONPs and subsequent release of Fe ions could be one ofprobable mechanism of action of IONPs [Singh N, et. al., Nano Rev. 2010:1:1-15]. Liu et al. and Bai et al. reported that the killing ofcancerous cells might be due the production of reactive oxygen speciesby IONPs or hyperthermia [Liu G, et. al., Small 2013; 446(9):1533-1545].However in other several studies it has been reported that the probablemechanisms of killing of cancer cells by IONPs could be due loss ofmembrane integrity, DNA damage, arrest of cell cycle, and cell apoptosis[Feng, Q, et. al., Sci. Rep. 2018; 8:2082; Liu, Y, et. al.,Nanotechnology 2014; 25 (449): 425101; Chen, J, et. al., Biomaterials.2015; 71: 168-177; and Palanisamy, S, et. al., Dalton Trans. 2019;2(48): 9490-9515]. It has been reported that the paramagnetic andsupermagnetic IONPs showed anticancer activity when appliednear-infrared or oscillating magnetic fields [Laurent S, et. al., AdvColloid Interface Sci. 2011; 166:8-23.]. Orel et al. reported that theantitumor effect of anticancer drug doxorubicin enhanced when complexwith magnetic Fe₃O₄ NPs [Orel V, et. al., Nanomedicine. 2015; 11:47-55].Several studies reported that IONPs has been widely applied in magnetictumor hyperthermia along with radiotherapy or chemotherapy for thetreatment of glioblastoma, glioma and prostate cancer [Van Landeghem FK, et. al., Biomaterials. 2009; 30:52-57; Silva A C, et. al., Int JNanomed. 2011; 6:591-603; Johannsen M, et. al., Int J Hyperthermia.2010; 26:790-795; and Maier-Hauff K, et. al., J Neurooncol. 2011;103:317-324]. Due to magnetic property and anticancer activity ofsynthesized L52E-γ-Fe₂O₃ NPs in this study, it has been suggested thatthe anticancer activity of IONPs can be further enhanced by applyingexternal magnetic field alone or in amalgamation with other cancertherapeutics drugs. However, future investigations on the exact killingmechanisms and safety profile of L52E-γ-Fe₂O₃ NPs on HCT-166 cells andother cancerous cell are warranted.

1. The method of claim 15, further comprising: making the iron oxidenanoparticles disinfectant composition by: mixing an iron precursorsolution comprising an iron (III) salt and water with an extractcomposition of the plant mixture to form a reaction mixture, heating thereaction mixture to form the iron oxide nanoparticles, and isolating theiron oxide nanoparticles; then mixing the iron oxide nanoparticles withthe saline solution to form the disinfectant composition.
 2. The methodof claim 1, further comprising: soaking the plant mixture in water in anamount of 1 g of plant mixture per 1 to 25 mL of water at 5 to 50° C.for 4 to 48 hours to form a plant suspension, and filtering the plantsuspension to form the extract composition.
 3. The method of claim 1,wherein the plant mixture comprises: 26 to 27.5 wt % Capparis, spinosa;26 to 27.5 wt % Cichorium intybus; 12.5 to 14 wt % Solanum nigrum; 6 to7 wt % Cassia occidentalis; 12.5 to 14 wt % Terminalia arjuna; 6 to 7 wt% Achillea millefolium; and 6 to 7 wt % Tamarix gallica.
 4. The methodof claim 1, wherein: the iron (III) salt is an iron (III) halide; andthe heating is performed at 40 to 80° C. for 15 to 180 minutes.
 5. Themethod of claim 1, wherein the reaction mixture has an iron (III)concentration of 0.25 to 1.25 mM and the extract is present in thereaction mixture in an amount of 24 to 120 mL per mmol of iron (III). 6.The method of claim 15, wherein the iron oxide nanoparticles comprisecrystalline γ-Fe₂O₃ by PXRD and have a mean particle size of 10 to 100nm by electron microscopy. 7-8. (canceled)
 9. The method of claim 15,wherein the iron oxide nanoparticles comprise crystalline γ-Fe₂O₃ byPXRD.
 10. The method of claim 15, wherein the iron nanoparticles have amean particle size of 10 to 100 nm by electron microscopy.
 11. Themethod of claim 15, wherein the iron nanoparticles have a saturationmagnetization of 17.5 to 27.5 emu/g and a coercivity less than 250 Oe at275 to 325 K.
 12. The method of claim 15, wherein the plant mixturecomprises: 26 to 27.5 wt % Capparis spinosa; 26 to 27.5 wt % Cichoriumintybus; 12.5 to 14 wt % Solanum nigrum; 6 to 7 wt % Cassiaoccidentalis; 12.5 to 14 wt % Terminalia arjuna; 6 to 7 wt % Achilleamillefolium; and 6 to 7 wt % Tamarix gallica.
 13. The method of claim15, wherein the extract composition comprises at least three selectedfrom the group consisting of n-hexadecanoic acid,(Z,Z)-9,12-octadecadienoic acid, (Z)-9-octadecenoic acid, octadecanoicacid, (Z)-3-(pentadec-8-en-1-yl)phenol, piperine,2-(hydroxymethyl)-2-nitro-1,3-propanediol, and tetradecanoic acid. 14.The method of claim 13, wherein the extract composition furthercomprises at least one selected from the group consisting of quercetin,kaempferol, cappariloside A, capparine A, capparine B, capparisine A,capparisine B, capparisine C, lactucin, lactucopicrin, aesculetin,aesculin, cichoriin, umbelliferone, scopoletin, 6,7-dihydrocoumarin,solasodine, solanine, emodin, cassiollin, cassia occidentanol I, cassiaoccidentanol II, arjunin, arjunic acid, arjungenin, arjunetin, arjunone,arjunoside I, arjunoside II, arjunoside III, arjunoside IV, archilletin,achilleine, apigenin, luteolin, tamarixin, tamarixetin,4-methylcoumarin, and troupin.
 15. A method for treating a biologicallycontaminated surface, comprising: contacting the biologicallycontaminated surface with a disinfectant composition, wherein thebiologically contaminated surface comprises one or more of a bacteriaand a fungus; exposing the bacteria and/or the fungus to thedisinfectant composition, wherein the disinfectant composition comprisesiron oxide nanoparticles, and a saline dispersion medium; wherein theiron oxide nanoparticles are stabilized with an extract of a plantmixture comprising Capparis spinosa, Cichorium intybus, Solanum nigrum,Cassia occidentalis, Terminalia etrjuna, Achillea millefolium, andTamarix gallica, and the iron oxide nanoparticles are suspended in thedispersion medium.
 16. The method of claim 15, wherein the bacteriaand/or the fungus comprises Pseudomonas aeruginosa (MDR-PA),Methicillin-resistant Staphylococcus aureus (MRSA) or Candida albicans.17. (canceled)
 18. The method of claim 16, wherein the iron oxidenanoparticles have a minimum inhibitory concentration (MIC) for P.aeruginosa of 0.60 to 1.5 mg iron oxide nanoparticles per mL, a MIC forS. aureus of 0.9 to 2.45 mg iron oxide nanoparticles per mL, and a MICfor C. albicans of 1.30 to 2.85 mg iron oxide nanoparticles per mL.19-20. (canceled)